Glue composition for lung volume reduction

ABSTRACT

The present invention relates to methods and compositions for sealing localized regions of damaged lung tissue to reduce overall lung volume. The glue compositions provide a glue featuring an adhering moiety coupled to one or more other moieties including, for example, a cross-linkable moiety and/or one other adhering moiety. The methods and compositions of the invention find use, for example, in treating pulmonary conditions, such as emphysema.

RELATED APPLICATIONS

This application claims priority to provisional applications U.S.60/580,444, entitled “Targeting Damaged Lung Tissue,” filed Jun. 16,2004; U.S. 60/586,932, entitled “Targeting Damaged Lung Tissue UsingVarious Formulations,” filed Jul. 8, 2004; and U.S. 60/586,950, entitled“Lung Volume Reduction Using Glue Composition,” filed Jul. 8, 2004, eachof which is incorporated herein in its entirety.

BACKGROUND OF THE INVENTION

Pulmonary conditions affect millions of Americans and many moreindividuals worldwide. Chronic obstructive pulmonary disease (COPD), forexample, including emphysema, asthma, bronchiectais and chronicbronchitis, is one of the most common chronic conditions and the fourthleading cause of death in the United States. While various environmentaland genetic factors may contribute to COPD, cigarette smoking is theprimary cause. Cigarette smoke can trigger inflammatory responses withinthe lungs, activating elastase, cathepsin G, and matrixmetalloproteinases (MMPs). These enzymes are proteases that result inprogressive destruction of the elastic tissue of the lungs, reducing theelasticity and lung recoil required for exhalation. Damaged alveolarwalls can eventually rupture to form inelastic “blebs.” Emphysema, forexample, is characterized by abnormal enlargement of alveolar airspacesdistal to terminal bronchioles and destruction of airspace parenchymaresulting in such “blebs.”

Current treatments are wanting. Treatment of pulmonary conditions ofteninvolves control and management rather than a cure for the disease. Withemphysema, for example, treatment can involve cessation of smoking,exercise programs, medications that help open constricted airways,anti-inflammatory medications, oxygen therapy, placement of one-wayvalves, and lung volume reduction surgery (LVRS). LVRS involves surgicalremoval of damaged, over-inflated lung tissue to free up space for theexpansion of remaining non-damaged tissue. This technique, however,requires invasive procedures and benefits tend to decline over time.Further, treatments using one-way valves have not proved satisfactory.Thus, there remains a need for improved methods for treating pulmonaryconditions, such as emphysema.

The present invention provides methods and compositions directedthereto. Other methods and compositions directed thereto are provided inU.S. nonprovisional applications entitled “Targeting Damaged Lung TissueUsing Compositions,” filed Dec. 8, 2004; “Targeting Damaged LungTissue,” filed Dec. 8, 2004; “Targeting Sites of Damaged Lung TissueUsing Composition,” filed Dec. 8, 2004; “Targeting Sites of Damaged LungTissue,” filed Dec. 8, 2004; “Imaging Damaged Lung Tissue UsingCompositions,” filed Dec. 8, 2004; “Imaging Damaged Lung Tissue,” filedDec. 8, 2004; “Glue Compositions for Lung Volume Reduction,” filed Dec.8, 2004; “Lung Volume Reduction Using Glue Compositions,” filed Dec. 8,2004; and “Lung Volume Reduction Using Glue Composition,” filed Dec. 8,2004, each of which is herein incorporated in its entirety.

BRIEF SUMMARY OF INVENTION

One aspect of the invention relates to a glue composition comprising afirst adhering moiety and a second adhering moiety where the adheringmoieties are coupled and where the adhering moieties adhere differentsites of lung tissue. In some embodiments, the lung tissue comprisesepithelial lining fluid. In some embodiments, the different sitescomprise different sites within an enlarged air space. In someembodiments, the first and second adhering moieties are the same. Insome embodiments, the first and second adhering moieties are different.

In some embodiments, the adhering moieties are coupled via a chemicallinker. In some embodiments, the chemical linker comprises twofunctional groups. In some embodiments, at least one of the functionalgroups is a hydroxyl group, a carboxyl group, an ester group, an aminegroup, or a lysine group. In some embodiments, at least one of thefunctional groups is a cyano group, a thiol group, a cysteine group, acarbonyl group, an aldehyde group or a ketone group. In someembodiments, the adhering moieties are coupled as a fusion polypeptide.In some embodiments, the adhering moieties are coupled via a protein. Insome embodiments, the adhering moieties are coupled via an antibody. Insome embodiments, the glue composition does not comprise apolysaccharide or a carbohydrate moiety. In some embodiments, the gluecomposition does not comprise a mutant plasminogen activator-inhibitortype 1.

In some embodiments, the adhering moiety adheres a cell surface marker.In some embodiments, the adhering moiety adheres an ECM component. Insome embodiments, the first and/or second adhering moiety adhereselastase. In some embodiments, the first and/or second adhering moietyadheres neutropil elastase. In some embodiments, the first and/or secondadhering moiety comprises a protease inhibitor moiety. For example, insome embodiments, the first and/or second adhering moiety comprises analpha-1 antitrypsin moiety, for example, a recombinant alpha-1antitrypsin moiety. In some embodiments, the first and/or secondadhering moiety comprises an elafin moiety, for example, a recombinantelafin moiety. In some embodiments, the first and/or second adheringmoiety comprises a serpin moiety, for example, a recombinant serpinmoiety, a secretory leukoprotease inhibitor (SLP1) moiety, and/or arecombinant secretory leukoprotease inhibitor (SLP1) moiety. In someembodiments, the first and/or second adhering moiety adheres at leastone matrix metalloproteinase selected from MMP-1, MMP-2, MMP-3, MMP-4,MMP-5, MMP-6, MMP-7, MMP-8, and MMP-9. In some embodiments, the gluecomposition does not comprise a hyaluronic acid or a salt thereof. Insome embodiments, the first and/or second adhering moiety adheresdesmosine and/or isodesmosine. In some embodiments, the first and/orsecond adhering moiety adheres CD8 and/or CD4. In some embodiments, thefirst and/or second adhering moiety adheres a smoke-related moiety.

In some embodiments, the glue composition is less than 10 microns. Insome embodiments, the glue composition is less than 5 microns. In someembodiments, the glue composition is less than 1 micron.

In another aspect of the invention, the glue composition furthercomprises a cross-linkable moiety coupled to the first and/or secondadhering moieties.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 illustrates one embodiment of a method to reduce lung volumeusing a glue composition comprising a cross-linkable moiety coupled toan adhering moiety that adheres to lung tissue.

FIG. 2 illustrates one embodiment of a method to reduce lung volumeusing a glue composition comprising coupled adhering moieties thatadhere to different sites of lung tissue.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the present invention provides a glue compositioncomprising an adhering moiety that adheres lung tissue, including lungfluids, such as, for example, epithelial lining fluid. An adheringmoiety may adhere to lung tissue, for example, sites of non-diseased ornormal lung tissue, as well as sites of diseased and/or non-normal lungtissue that may be affected, have been affected, or are likely to beaffected by a pulmonary condition. An adhering moiety may bind, attach,or otherwise couple to lung tissue by covalent and/or non-covalentbinding. Examples of binding forces that may be useful in the presentinvention include, but are not limited to, covalent bonds, dipoleinteractions, electrostatic forces, hydrogen bonds, hydrophobicinteractions, ionic bonds, and/or van der Waals forces.

In some preferred embodiments, the adhering moiety adheres to a proteaseor other molecule and/or macromolecule present in lung tissue. Forexample, the adhering moiety may adhere a molecule and/or macromoleculefound attached, bound, coupled, complexed and/or otherwise associatedwith lung tissue. Binding, attachment, coupling, complexing and/orassociation may involve covalent and/or non-covalent interactions,including, e.g., dipole interactions, electrostatic forces, hydrogenbonds, hydrophobic interactions, ionic bonds, and/or van der Waalsforces.

In some embodiments, the adhering moiety may adhere a molecule and/ormacromolecule that is bound, attached, coupled, complexed and/orotherwise associated with a cell surface of lung tissue. In someembodiments, the molecule and/or macromolecule may be bound to a cellwall. In some embodiments, the molecule and/or macromolecule may becomplexed with a moiety that is itself bound to a cell wall. In someembodiments, the molecule and/or macromolecule may comprise a cellsurface marker. In still some embodiments, the molecule and/ormacromolecule may be found associated with the extra cellular matrix(ECM). For example, the molecule and/or macromolecule may comprise anECM component or may be associated with an EMC component of lung tissue.

In some embodiments, the adhering moiety may adhere a molecule and/ormacromolecule comprising at least one moiety selected from a proteinmoiety, a glycoprotein moiety, a lipoprotein moiety, a lipid moiety, aphospholipid moiety, a carbohydrate moiety, a nucleic acid moiety, amodified nucleic acid moiety, and/or a small molecule moiety, including,e.g., a cell surface marker comprising a glycoprotein moiety and/or anECM component comprising a protein moiety.

In some preferred embodiments, the adhering moiety adheres to elastase.The elastase may be bound to the cell wall and/or associated with theextracellular matrix of lung tissue. For example, elastase causesprogressive destruction of elastic fibers of lung tissues in somepulmonary conditions, e.g., emphysema, resulting in dilation and ruptureof distended alveoli to form characteristic “blebs.” Suki et al., “Onthe Progressive Nature of Emphysema, Pulmonary Perspective”, AmericanJournal of Respiratory and Critical Care Medicine, Vol. 168 pgs. 516-520(2003); Janoff et al., Am. Rev. Respir. Dis., Vol. 132 pgs. 417-433(1985); Senior and Kuhn, In Fishman (ed), Pulmonary Diseases andDisorders, 2d ed. New York, McGraw-Hill, p. 1209-1218 (1988). In somepreferred embodiments, the adhering moiety adheres to neutrophilelastase and/or neutrophils. In some preferred embodiments, the adheringmoiety adheres pancreatic and/or macrophage elastase. In some preferredembodiments, the adhering moiety adheres neutrophil proteinase 3 (Pr3).Pr3 is descried, for example, in Duranton et al., “Inhibition ofproteinase 3 by alpha-1 antitrypsin in vitro predicts very fastinhibition in vivo”, Am J Respir Cell Mol. Biol., Vol. 29 No. 1 pgs57-61 (2003).

For example, the adhering moiety may (or may not) comprise alpha-1antitrypsin, elafin, thypin (see, e.g., International Publication No. WO02/072769), and/or other serpin, e.g., PAI-1, PAI-2, SCCA-1, SCCA-2,secretory leukoprotease inhibitor SLP-1, (see, e.g., U.S. Pat. No.6,753,164), and/or other serpin-related proteins (e.g., as disclosed inU.S. Publication No. 2004/0126777); a recombinant form of any of theseand/or a moiety of any of these that retains the ability to adhere tolung tissue. In some embodiments, the adhering moiety may (or may not)comprise mucous proteinase inhibitor (MPI) that shows high affinity forbinding to elastase. Belorgey et al., “Effect of polynucleotides on theinhibition of neutrophil elastase by mucus proteinase inhibitor andalpha-1 proteinase inhibitor”, Biochemistry, Vol. 37 No. 46 pgs 16416-22(1998). Other adhering moieties that can adhere to elastase may also beused, such as inhibitors of elastase known in the art. See, e.g., Janoffet al., Am. Rev. Respir. Dis. Vol. 132 pgs 417-433 (1985); Zimmerman andPowers (1989), In Hornebeck (ed), Elastin and Elastases, vol II, BocaRaton, CRC Press, pgs 109-123; and Laurell and Eriksson Scand. J. Clin.Lab. Invest., Vol. 15 pgs 132-140 (1963). Other adhering moieties caninclude protease inhibitors of the inter-alpha trypsin inhibitor (ITI)family. The ITI protein family can be built up from differentcombinations of the polypeptides HC1, HC2, HC3 and bikunin, asdescribed, e.g., in Cuvelier et al., “Proteins of the inter-alphatrypsin inhibitor (ITI) family. A major role in the biology of theextracellular matrix”, Rev Mal Respir., Vol. 17 No. 2 pgs 437-46 (2000).

Alpha-1 antitrypsin useful for preparing an adhering moiety of thepresent invention may be obtained by any techniques known in the artand/or disclosed herein. For example, alpha-1 antitrypsin can beobtained by recombinant methods, as known in the art (e.g., recombinantalpha-1 antitrypsin from Novartis). Techniques for purifying alpha-1antitrypsin, e.g., from biological natural and/or recombinant sourcesare also known in the art. See, e.g., International Publication No. WO00/17227 and U.S. Pat. No. 4,656,254, which describes separating alpha-1antitrypsin from plasma.

In some preferred embodiments, the adhering moiety adheres to desmosineand/or isodesmosine. Desmosine and/or isodesmosine are amino acidsproduced as a result of damage to lung tissues, particularly damageinvolving destruction of elastin. Fragmented elastin, for example, ismetabolized to free desmosine or small peptides, which can be recoveredin the urine of the subject. See, e.g., Starcher B. C., “Lung Elastinand Matrix”, Chest, Vol. 117 pgs 229S-234S (2000). In animal models ofemphysema, for example, desmosine urine recovery can serve as a measureof lung damage. There are several micromethods for measuring desmosine,including, for example, enzyme linked immunosorbent assay (see, e.g.,Osakabe T. et al. “Comparison of ELISA and HPLC for the determination ofdesmosine and isodesmosine in aortic tissue elastin”, J. Clin Lab AnalVol. 9 pgs 293-296 (1995)); isotope dilution (see, e.g., Stone P. J. etal. “Measurement of urinary desmosine by isotope dilution and highperformance liquid chromatography”, Am Rev Respir Dis Vol. 144 pgs284-290 (1991)); high performance liquid chromatography (see, e.g.,Covault H. P. et al. “Liquid-chromatographic measurement of elastin”,Clin Chem. Vol. 28 pgs 1465-1468 (1982)); and/or radioimmunoassay (see,e.g., Starcher B. “A role for neutrophil elastase in the progression ofsolar elastosis”, Connect Tissue Res Vol. 31 pgs 133-140 (1995)).

In some preferred embodiments, the adhering moiety adheres to cathepsin,e.g., cathepsin G, which can be produced by inflammatory cells in thepathogenesis of COPD. In some embodiments, the adhering moiety adheresother cysteine proteinases. In some embodiments the adhering moietyadheres cathepsins L, S, and K. In some embodiments, the adhering moietyadheres RGS2, which accumulates at sites of macrophage activation, e.g.,in activated-macrophage-related disorders, including emphysema. See,e.g., EP 1378518. In some embodiments, the adhering moiety adheres toalveolar macrophages. In some embodiments, the adhering moiety adheresto eosinophils. In some embodiments, the adhering moiety adheres totumor necrosis factor-α. In some embodiments, the adhering moietyadheres to kallikrenin.

In some preferred embodiments, the adhering moiety adheres acollagenase. The presence of collagenase activity may be detected, forexample, by released components, e.g., amino acids, known to occur incollagen, e.g., hydroxyproline and/or hydroxylysine.

Examples of collagenases include, e.g., one or more metalloproteinases.Metalloproteinases include, e.g., MMP-1 (interstitial collagenase orcollagenase-1), MMP-2 (gelatinase-A or 72 kD gelatinase), MMP-3(transin, human fibroblast stromelysin or stromelysin-1), MMP-4, MMP-5,MMP-6, MMP-7 (matrilysin), MMP-8 (collagenase-2 or neutrophilcollagenase), MMP-9 (gelatinase B or 92 kD gelatinase), MMP-10(stromelysin II), MMP-11 (stromelysin III), MMP-12 (macrophasemetalloelastase), and/or MMP-13 (collagenase-3) and as well asmetalloproteinase ADAM 2222 (see, e.g., U.S. Publication No.2003/0194797). Metalloproteinases (also referred to as metalloproteasesin the art) have been described, e.g., U.S. Publication No.2003/0199440; U.S. Publication No. 2004/0048302; U.S. Publication No.2004/0043407; U.S. Publication No. 2004/019479; and InternationalPublication No. WO 02/072751. For example, an adhering moiety comprisingan ilomastat moiety may be used. See, e.g., International PublicationNo. WO 2004/052236.

In some embodiments, the glue composition does not comprise apolysaccharide or carbohydrate moiety, e.g., in some embodiments, theglue composition does not comprise hyaluronic acid or a salt thereof;and in some embodiments, the glue composition does not comprise dextranor glycosaminoglycan. In some embodiments, the glue composition does notcomprise a polysaccharide or carbohydrate moiety that binds to elasticfibers. In some embodiments, the glue composition does not comprise anantibody. In some embodiments, the glue composition does not comprise alung membrane dipeptidase-binding molecule, e.g., in some embodiments,the glue composition may not adhere to lung membrane dipeptidase, and insome embodiments the glue composition may not comprise GFE-1 peptide.See, e.g., Ruoslahti et al., “Membrane dipeptidase is the receptor for alung-targeting peptide identified by in vivo phage display”, J BiolChem. Vol. 274 No. 17 pgs 11593-8 (1999) and U.S. Pat. No. 6,784,153.

Also, in some preferred embodiments, the adhering moiety adheres to CD8and/or CD4, CD8 lymphocytes and/or CD4 lymphocytes, and/or interleukin 8(see, e.g., U.S. Publication No. 2003/0232048). In some embodiments, theadhering moiety adheres to mitogen-activated protein kinase(International Publication No. WO 03/064639). In some embodiments, theadhering moiety may (or may not) adhere to CIRL-2 homologs (see, e.g.,International Publication No. WO 2004/031235). In still someembodiments, the adhering moiety may (or may not) comprise an antibodyand/or binding fragment thereof that adheres to lung tissue. Forexample, the adhering moiety may comprise a COPD-related human Igderived protein, discussed e.g. in International Publication No. WO02/072788 and/or U.S. Publication No. 2003/0017150, which can bind COPDrelated proteins. In yet another example, the adhering moiety maycomprise an antibody to secreted protein HCEJQ69 (see, e.g., U.S. Pat.No. 6,774,216).

Preferred adhering moieties of the present invention comprise biologicalmoieties, such as proteins or polypeptides, which can adhere to lungtissue, and can include naturally-occurring protease inhibitors, such asalpha-1 antitrypsin and/or mutants thereof and/or fragments thereof, aswell as other protease inhibitor moieties. As well as alpha-1antitrypsin, other naturally-occurring inhibitors of elastase may alsobe used as preferred adhering moieties of the present invention,including, e.g., monocyte elastase inhibitor and variants thereof (see,e.g., International Publication No. WO 96/10418; U.S. Pat. No.5,827,672; and U.S. Pat. No. 5,663,299); as well as tissue inhibitors ofmetalloproteinases (TIMPs), such as TIMP-1, TIMP-2, TIMP-3, and TIMP-4.

In more preferred embodiments, the adhering moiety is modified such thatit binds to lung tissue irreversibly, substantially irreversibly, or atleast with a high binding constant, e.g., to resist dissociation for adesired period of time. Adhering moieties may be selected and/ordeveloped to increase binding affinity for lung tissue. For example,alpha-1 antitrypsin may be mutated by random and/or directed synthesis,to engineer mutants with higher binding constants for elastase. Forexample, in some preferred embodiments, the adhering moiety has a K_(i)value against a component of lung tissue of less than about 200 nM, lessthan about 150 nM, less than about 100 nM, or less than about 75 nM. Insome preferred embodiments, the adhering moiety has a K; value against acomponent of lung tissue of more than about 50 nM, more than about 25nM, more than about 20 nM, more than about 15 nM, more than about 10 nM,more than about 5 nM, more than about 3 nM, or more than about 1 nM. Insome preferred embodiments, the adhering moiety binds a component oflung tissue with a K_(D) less than about 10⁻⁸ M, less than about 10⁻⁹ M,less than about 10⁻¹⁰ M, less than about 10⁻¹¹ M, less than about 10⁻¹²M, less than about 10⁻¹³ M, or less than about 10⁻¹⁴ M.

Other non-naturally occurring protease inhibitors that may (or may not)be used as an adhering moiety of the present invention includeinhibitors of neutrophil elastase (e.g., methyl ketone derivatives);inhibitors of macrophage metalloproteinase (e.g., RS113456 andinhibitors discussed in U.S. Publication No. 2003/0199440); Cathepsin Ginhibitors (e.g., LEX-032 (Sparta)); various elastase inhibitors (e.g.ABT-491 (Abbot)); inhibiting compositions (e.g., as disclosed in U.S.Publication No. 2003/0199440 and International Publication No. WO03/090682, including lipase inhibitors and phospholipase inhibitors);protease inhibitor compositions (e.g., as disclosed in InternationalPublication No. WO 2004/045634); Erdosteine (Edmond Pharma), FK-706(Fujisawa), GW-311616 (Glaxo-Wellcome), Midesteine (Medea); a mutantplasminogen activator-inhibitor type 1 (see, e.g., U.S. Publication No.2003/0216321); an N-substituted azetidinone (see, e.g., EP 0529719);peptidyl carbamates (e.g., U.S. Pat. No. 5,008,245 and/or EP 0367415);SR-268794 (Sanoti) and/or SYN-1134 (Syn. Pharm.); other proteinaseinhibitors (e.g., CMP-777 (Dupont)); heteroaryl aminoguanidines andalkoxyguanidines (see, e.g., U.S. 2004/0106633 and EP 1070049); as wellas ON-elastase inhibitors (e.g., NX-21909 (Gilead)); and several HNEinhibitors (e.g., CE-1037 (Cortech/United Ther), CE-2000 series(Cortech/Ono), EPI-HNE-4 (Dyax), EPI-HNE-1 (Protein Engineer),MDL-101146 (HMR), Ono-5046 (Ono), SPAAT (UAB Res. Found.), WIN-63759(Sterling Winthrop), ZD-8321 (AstraZeneca), and/or ZD-0892(AstraZeneca)). Adhering moieties may (or may not) also includeinhibitors and/or antibodies of any lung tissue components describedherein, as well as inhibitors and/or antibodies of proteins described inInternational Publication No. WO 03/010327; as well as inhibitors and/orantibodies of eosinophil serine protease 1-like enzymes described inU.S. Publication No. 2003/0224430 and/or other serine proteases, e.g.,described in International Publication No. WO 2004/053117; as well asinhibitor and/or antibodies of transmembrane serine proteases, e.g., asdiscussed in U.S. Pat. No. 6,734,006; as well as inhibitors and/orantibodies of esterase described in International Publication No. WO04/020620. As used herein, “antibodies” includes binding fragmentsthereof.

In some preferred embodiments, the adhering moiety comprises a compound,such as a small molecule compound, that adheres lung tissue or acomponent thereof. Such compounds can be obtained, for example, vialigand screening methods, as known in the art. For example, a biologicalsample or a defined candidate moiety can be brought into contact with acomponent of lung tissue, for example purified and/or recombinantelastase, or fragments thereof, as well as a component isolated and/orpurified from epithelial lining fluid. The candidate moiety may belabeled with a detectable label, such as a fluorescent, radioactive,and/or an enzymatic tag and allowed to contact the lung tissue componentthat may be immobilized, e.g., under conditions that permit binding.After removing unbound moieties, bound moiety can be detected usingappropriate methods as known in the art.

Candidate moieties that can be assayed for adhering lung tissue for usein the present invention are not limited. For example, such candidatemoieties can be obtained from a wide variety of sources includinglibraries of synthetic, semi-synthetic and/or natural substances. Randomand/or directed synthesis can be used, for example, to generate a widevariety of organic compounds and biomolecules, including randomizedoligonucleotides and oligopeptides. With respect to natural compounds,libraries form bacterial, fungal, plant and animal extracts areavailable and/or can be readily produced. Further, natural,semi-synthetically, and/or synthetically produced libraries can bemodified through conventional chemical, physical, recombinant, and/orbiochemical techniques to produce combinatorial libraries. Also, knownpharmaceutical or pharmacological agents may be modified by directed orrandom chemical modifications, including, for example, acylation,amidification, alkylation, and/or esterification to produce structuralanalogs.

Candidate moieties may include natural, synthetic and/or semi-syntheticorganic compounds, macromolecules of biological origin, such aspolypeptides, peptides, polysaccharides, glycoproteins, lipoproteins,fatty acids, and/or fragments thereof; and/or drugs or small molecules,such as molecules generated through combinatorial chemistry approaches.Further, when the candidate moiety comprises a peptide or polypeptide,the candidate moiety may be expressed by a phage clone belonging to aphage-based random peptide library (see, e.g., Parmley and Smith, GeneVol. 73 pgs 305-318 (1988); Oldenburg et al., Proc. Natl. Acad. Sci. USAVol. 89 pgs 5393-5397 (1992); Valadon et al., J. Mol. Biol., Vol. 261pgs 11-22 (1996); Westerink, Proc. Natl. Acad. Sci. USA., Vol. 92 pgs4021-4025 (1995); and Felici et al., J. Mol. Biol., Vol. 222 pgs301-310) (1991); and/or the candidate moiety may be expressed from acDNA cloned in a vector for performing a two-hybrid screening assay(U.S. Pat. Nos. 5,667,973 and 5,283,173; Harper et al., Cell, Vol. 75pgs 805-816 (1993); Cho et al., Proc. Natl. Acad. Sci. USA, Vol. 95(7)pgs 3752-3757 (1998); and Fromont-Racine et al., Nature Genetics, Vol.16(3) pgs 277-282 (1997).

The adhering moiety may also adhere to a smoke-related moiety. Forexample, the adhering moiety may bind to cigarette smoke particles, tar,tobacco, and/or other smoke-related residues, such as Cadmium. Further,it is to be understood that the adhering moiety may adhere one of morecomponents of lung tissue and/or smoke-related moieties, including anycombination of proteases disclosed herein, as well as one or moreproteases and/or one or more smoke-related moieties.

In some embodiments, the adhering moiety may adhere to modifiedpolypeptides. For example, members of the G-protein coupled receptor(GPCR) family, e.g., RAI-3 are modified, e.g., phosphorylated, and/orassociated with tyrosine phosphorylated activation complexes followingexposure to cigarette smoke. See, e.g., International Publication No. WO04/001060 and/or U.S. Publication No. 2004/0121362. In some embodimentsof the present invention, an adhering moiety may be used that adheres tosuch modified proteins and/or protein complexes. Such adhering moietiesmay (or may not) include modulators of RAI-3, as described in U.S.Publication No. 2004/0121362. In still some embodiments, an adheringmoiety may (or may not) be used that adheres polypeptides associatedwith the NF-kB pathway that are found in lung tissue, e.g., as describedin U.S. Publication No. 2004/0086896.

The adhering moiety may also adhere moieties that inhibit the productionof elastic and/or connective tissue proteins. Such moieties may include,e.g., moieties that inhibit fibroblast proliferation and/or that inhibitprocollagen production and/or that inhibit proteoglycan synthesis,preferably moieties that inhibit synthesis of the majormatrix-associated proteoglycans, such as versican, decorin, and/or largeheparan sulfate proteoglycans. “Inhibiting” and its various grammaticalconjugations can mean reducing a biological process, e.g., reducingsynthesis of a connective tissue component, by an amount compared withthe occurrence of the process in the absence (or in the presence oflower levels) of the inhibiting moiety. In some embodiments, the amountmay be reduced by at least about 10%, at least about 20%, at least about30%, at least about 40%, or at least about 50%. In some embodiments, theamount may be reduced by less than about 60%, less than about 70%, lessthan about 80%, less than about 90%, or less than about 95%.“Inhibiting” and its various grammatical conjugations need not meancompletely inhibiting a biological process, e.g., it need not meaninhibiting synthesis of a connective tissue component to negligibleand/or non-detectable levels. Moieties that can inhibit proteoglycansynthesis include, for example, Cadmium. See, e.g., Chambers et al.,“Cadmium inhibits proteoglycan and procollagen production by cultureshuman lung fibroblasts,” Am. J. Respir. Cell Mol. Biol., Vol. 19 No. 3pgs 498-506 (1998). Other moieties may include lead, aldehydes and/orsilicates. Fujiwara, “Cell biological study on abnormal proteoglycansynthesis in vascular cell exposed to heavy metals,” Journal of HealthScience, Vol. 50 No. 3 pgs 197-204 (2004). The adhering moiety may alsoadhere to moieties that impair the repair of elastic and/or connectivetissues of the lungs.

In some aspects of the present invention, a glue composition comprisingan adhering moiety also comprises a cross-linkable moiety coupledthereto. The cross-linkable moiety can be any moiety that facilitateslinkage between more than one cross-linkable moieties, preferablybetween cross-linkable moieties coupled to adhering moieties binding tolung tissue at different sites. Cross-linkable moieties can include, forexample, a hydroxyl group, carboxyl group, ester group, cyano group,thiol group (including e.g., a cysteine group), carbonyl group, aldehydegroup, ketone group, primary amine group, and/or secondary amine group,as well as a lysine group.

In some embodiments, the cross-linkable moiety comprises any other aminegroups, a sulfide group, a carbonyl group (e.g., α-halocarbonyl groupand/or a, fi-unsaturated carbonyl group), a cyanate group (e.g.,isothiocyanate group), a carboxylate group (e.g., an acetate group suchas a-haloacetate group), a hydrazine group, and/or a biotin group, See,e.g., US Publication No. 2002/0071843.

In some embodiments, the cross-linkable moiety can comprise fibrinogenand/or fibrin. Fibrinogen can be converted to fibrin, which ispolymerized in a cross-linking reaction. In some embodiments, thecross-linkable moiety can comprise other protein and/or proteinaceousmaterials, e.g., proteinaceous materials comprising albumin (bovine orhuman), collagen, PEI, oleic acid, chitin and/or chitosan, as well asany of those described in U.S. Pat. No. 5,385,606, U.S. Pat. No.5,583,114, U.S. Pat. No. 6,310,036, U.S. Pat. No. 6,329,337, and/or U.S.Pat. No. 6,372,229. In some embodiments, more than one type ofcross-linkable moiety may be coupled to a given adhering moiety or maybe coupled to a number of adhering moieties used in combination, e.g.,in one administration or in a number of successive administrations.Those of skill in the art will recognize other suitable cross-linkablemoieties that may be used in the practice of the instant invention,including, for example, any biocompatible cross-linkable moiety that canform a biocompatible cross-linked product.

The adhering moiety may be coupled to the cross-linkable moiety by anytechniques and/or approaches known in the art, described herein, and/oras can be developed by those of skill in the art. For example, couplingmethods include, but are not limited to the use of bifunctional linkers,amide formation, imine formation, carbodiimide condensation, disulfidebond formation, and/or use of a specific binding pair e.g., using abiotin-avidin interaction. These and other methods known in the art maybe found, e.g., in Hermanson, “Bioconjugate Techniques,” Academic Press:New York, 1996; and S. S. Wong, “Chemistry of Protein Conjugation andCross-linking,” CRC Press, 1993.

In preferred embodiments, the cross-linkable moiety is coupled to theadhering moiety in such a way so as not, to interfere with the abilityof the adhering moiety to adhere to lung tissue. For example thecross-linkable moiety can be attached to an alpha-1 antitrypsin moietyat one or more sites that do not modify the conformation or folding ofthe alpha-1 antitrypsin, or do not modify the conformation or folding ofregions of alpha-1 antitrypsin necessary and/or involved in adhering tosites of lung tissue, e.g. adhering to elastase present in lung tissue.For example, without being limited to a given hypothesis or mode ofaction, the active inhibitory site of alpha-1 antitrypsin is foundaround Ser358 of the polypeptide, e.g., forming a pseudo-irreversibleequimolar complex with neutrophil elastase. See, e.g., Sifers et al.,“Genetic Control of Human Alpha-1 Antitrypsin”, Mol. Biol. Med., Vol. 6pgs 127-135 (1989). In some preferred embodiments, a cross-linkablemoiety can be attached to an alpha-1 antitrypsin moiety at a site otherthan around its Ser358 inhibitory site. Similarly, in some embodiments,without being limited to a given hypothesis or mode of action, across-linkable moiety can be attached to a serpin moiety at a site otherthan certain regions known to be involved in attaching to a protease,which include, for example, the hinge, breach, shutter, and gate regionsof serpins. Irving et al., Genome Res Vol. 10 pgs 1845-64 (2000).Similarly, in some embodiments, without being limited to a givenhypothesis or mode of action, a cross-linkable moiety can be attached toa monocyte elastase inhibitor moiety at a site other than a cysteineresidue of the inhibitor involved in interacting with elastase and/orproteinase 3 and/or cathepsin G. See, e.g., International. PublicationWO 96/10418; and U.S. Pat. No. 5,827,672.

In some embodiments, the cross-linkable moiety may be chemically boundto the adhering moiety, e.g., a carboxyl group covalently attached toone or more sites of alpha-1 antitrypsin. In some embodiments, thecross-linkable moiety may be chemically bound to a moiety that is itselfchemically bound to the adhering moiety, indirectly coupling thecross-linkable and adhering moieties.

In preferred embodiments, the size of the glue composition comprising anadhering moiety coupled to a cross-linkable moiety is not so large as toprevent access of the glue composition to lung tissue within enlargedalveoli distal to a terminal bronchiole. For example, the size of theglue composition comprising an adhering moiety coupled to across-linkable moiety is preferably less than about 10 microns, lessthan about 8 microns, less than about 5 microns, less than about 3microns, less than about 2 microns, or less than about 1 micron.“Enlarged alveolus” as used herein refers to an alveolus that is largerthan the average alveolus that is not affected by a pulmonary condition,or that is affected to a lesser extent. For example, an enlargedalveolus may be at least about 5%, at least about 10%, at least about20%, at least about 50%, at least about 100%, or at least about 150% thesize of an average alveolus.

Another aspect of the present invention relates to a glue compositioncomprising a first adhering moiety and a second adhering moiety whereinsaid adhering moieties are coupled and wherein said adhering moietiesadhere to different sites of lung tissue. In preferred embodiments, thedifferent sites comprise different sites within an enlarged air space,e.g., within alveolar walls of an over-inflated alveolus distal to aterminal bronchiole, as characteristic of some pulmonary conditions,including emphysema. The first and second adhering moieties may be thesame or different.

Further, it is to be understood that any plural number of adheringmoieties may be used, i.e., the present invention also contemplates aglue composition comprising any plural number of coupled adheringmoieties, that may each be the same or different, or some may be thesame while others are different. For example, in a glue compositioncomprising three coupled adhering moieties, the first adhering moietymay be coupled to the second adhering moiety, which is coupled to athird adhering moiety. The first and third moieties may or may not bedirectly coupled to each other. In some embodiments, the three adheringmoieties may be coupled to a moiety without being directly coupled toeach other. The three moieties may all be the same or different, or twomay be the same with the third is different. Each adhering moiety mayadhere to the same of different components in lung tissue, preferablyadhering at different sites within an enlarged air space, e.g., withinalveolar walls of an over-inflated alveolus distal to a terminalbronchiole.

The adhering moieties may be coupled by any techniques and/or approachesknown in the art, described herein, and/or as can be developed by thoseof skill in the art. In some embodiments, coupling may involve covalentbonds, dipole interactions, electrostatic forces, hydrogen bonds,hydrophobic interactions, ionic bonds, van der Waals forces, and/orother bonds that can couple adhering moieties. For example, in someembodiments, adhering moieties are coupled via a coupling moiety, e.g.,a chemical linker. Any chemical linker may be used, including, e.g., analiphatic group covalently linking the adhering moieties. For example, achemical linker useful in this invention may comprise two (or more)functional groups, where each of the functional groups can be chemicallybonded to an adhering moiety, serving to couple the adhering moieties.Examples of functional groups include, e.g., a hydroxyl group, acarboxyl group, an ester group, a cyano group, a thiol group, a cysteinegroup, a carbonyl group, an aldehyde group, a ketone group, and/or anamine group, as well as a lysine group. Other function groups include acyanate group (e.g., isothiocyanate) and/or a carboxylate group (e.g.,an acetate group such as a-haloacetate).

Other coupling techniques may also be used. For example, dimers and/ormultimers of adhering moieties may be prepared using cross-linkingtechniques so that the adhering moieties are pre-cross-linked, e.g.,forming one or more cross-links between cysteine residues of peptideand/or polypeptide adhering moieties. Linker length optimizationtechniques may also be used (see, e.g., U.S. Pat. No. 5,478,925), foruse in the present invention.

In some embodiments, adhering moieties are coupled as a fusionpolypeptide. For example, where the adhering moieties are peptidesand/or polypeptides, two or more adhering moieties may be joined by apolypeptide linker as the coupling moiety, to form a fusion polypeptideor fusion protein. A fusion protein may be generated in various ways,including, e.g., chemical coupling and co-translation. In some preferredembodiments, adhering moieties are recombinantly expressed as a fusionproduct from a recombinant nucleic acid molecule, where the adheringmoieties are linked, e.g., by one or more intervening amino acids,according to techniques known in the art. See, e.g., Francis, “Focus onGrowth Factors”, Vol. 3 pgs 4-10 (Mediscript, London) (1992). Fusionproteins may also be made using other techniques known in the art, e.g.,techniques used to create adzymes, which comprise an address bindingsite conjugated to a catalytic domain (e.g., as described in U.S.Publication No. 2004/0081648 and in U.S. Publication No. 2004/0081648);and/or by covalent linking (e.g., via disulfide bonds) between at leastone amino acid of each coupled adhering moiety (e.g., as descried inU.S. Publication No. 2004/0087778).

In some embodiments, the adhering moieties are coupled via a protein,e.g., an antibody and/or a binging fragment thereof. In someembodiments, liposomes may be prepared that comprise a plural number ofadhering moieties.

In some preferred embodiments, the adhering moieties are coupled in sucha way so as not to interfere with the ability of the adhering moiety toadhere lung tissue. For example two (or more) alpha-1 antitrypsinmoieties can be coupled to each other at sites that do not modify theconformation or folding of the alpha-1 antitrypsin moieties, or do notmodify the conformation or folding of regions of the alpha-1 antitrypsinmoieties necessary and/or involved in adhering to sites of lung tissue,e.g. adhering to elastase present in lung tissue. For example, withoutbeing limited to a given hypothesis or mode of action, the activeinhibitory site of alpha-1 antitrypsin is found around Ser358 of thepolypeptide, e.g., forming a pseudo-irreversible equimolar complex withneutrophil elastase. See, e.g., Sifers et al., “Genetic Control of HumanAlpha-1 Antitrypsin”, Mol. Biol. Med., Vol. 6 pgs 127-135 (1989). Insome preferred embodiments, alpha-1 antitrysin moieties may be coupledto each other or other adhering moieties at sites other than aroundtheir Ser358 inhibitory sites. Similarly, in some embodiments, withoutbeing limited to a given hypothesis or mode of action, serpin moietiesmay be coupled to each other or other adhering moieties at sites otherthan certain regions known to be involved in attaching to protease,which include, for example, the hinge, breach, shutter, and gate regionsof serpins. Irving et al., Genome Res Vol. 10 pgs 1845-64 (2000). Someserpins, for example, contain a reactive center loop (RCL) involved ininhibition where a stable complex can be formed between the protease anda cleaved form of the serpin. Attachment via sites other than the RCLregions of serpin moieties is preferred in some embodiments. Similarly,in some embodiments, without being limited to a given hypothesis or modeof action, monocyte elastase inhibitor moieties can be coupled to eachother or other adhering moieties at a site other than a cysteine residueof the inhibitor involved in interacting with elastase and/or proteinase3 and/or cathepsin G. See, e.g., International Publication WO 96/10418and U.S. Pat. No. 5,827,672.

In preferred embodiments, the size of the glue composition comprisingtwo (or more) coupled adhering moieties is not so large as to preventaccess of the glue composition to sites of lung tissue within enlargedair spaces distal to a terminal bronchiole. For example, the size of theglue composition comprising two (or more) adhering moieties ispreferably less than about 10 microns, less than about 8 microns, lessthan about 5 microns, less than about 3 microns, less than about 2microns, or less than about 1 micron.

Coupling of the adhering moieties can keep the adhering moieties inclose or relatively close physical proximity. For example, in somepreferred embodiments a chemical linker may be used that comprises analiphatic group of at least about 2 carbon atoms, at least about 5carbon atoms, at least about 10 carbon atoms, or at least about 12carbon atoms. In some preferred embodiments, a chemical linker thatcomprises an aliphatic group of less than about 30 carbon atoms, lessthan about 20 carbon atoms, or less than about 15 carbon atoms can beused. In some preferred embodiments, a polypeptide linker can be usedthat comprises at least about one amino acid, at least about 3 aminoacids, or at least about 5 amino acids. In some preferred embodiments, apolypeptide linker that comprises less than about 12 amino acids, lessthan about 10 amino acids, or less than about 5 amino acids can be used.

Further, it is to be understood that a glue composition comprising two(or more) coupled adhering moieties may further comprise a coupled ornot coupled cross-linkable moiety.

Formulation, Routes of Administration, and Effective Doses

The glue compositions useful in the practice of the present inventioncan be delivered to a subject using a number of routes or modes ofadministration. The adhering moieties, cross-linkable moieties,cross-linking activating moieties, imaging moieties and/or othermoieties and/or agents may be delivered per se or as pharmaceuticallyacceptable salts thereof. The term “pharmaceutically acceptable salt”means those salts which retain the biological effectiveness and desiredproperties of the moieties and/or agents of the present invention, andwhich are not biologically or otherwise undesirable. Such salts includesalts with inorganic or organic acids, such as hydrochloric acid,hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid,methanesulfonic acid, p-toluenesulfonic acid, acetic acid, fumaric acid,succinic acid, lactic acid, mandelic acid, malic acid, citric acid,tartaric acid or maleic acid. In addition, if the moiety contains acarboxyl group or other acidic group, it may be converted into apharmaceutically acceptable addition salt with inorganic or organicbases. Examples of suitable bases include sodium hydroxide, potassiumhydroxide, ammonia, cyclohexylamine, dicyclohexyl-amine, ethanolamine,diethanolamine and triethanolamine.

The adhering, cross-linkable, cross-linking activating, imaging moietiesand/or other moieties and/or agents, or pharmaceutically acceptablesalts thereof, can be formulated with a pharmaceutically acceptablecarrier for administration to a subject in need thereof.“Pharmaceutically acceptable carriers” are well known in thepharmaceutical art, described, for example, in Remington'sPharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).Suitable carriers include, for example; carriers like alcohol, DMSO,saline solution, and/or water. Pharmaceutical compositions for use inaccordance with the present invention may be formulated in conventionalmanner using one or more physiologically acceptable carriers comprisingexcipients and/or auxiliaries, which facilitate processing of the activemoieties into preparations that can be used pharmaceutically. Properformulation is dependent upon the route of administration chosen.

In some embodiments, the glue compositions of the invention aredissolved in a suitable solvent, such as sterile water or PBS, and thendried to remove the solvent and produce a powder. Drying can be carriedout in such as way as to retain the desired properties of the gluecompositions, for example the capability of an adhering moiety to adherelung tissue. For example, vacuum concentration, spray drying, opendrying, freeze-drying, and the like, can be used. The residue obtainedcan then be ground and/or further micronized.

In some preferred embodiments, the adhering, cross-linkable,cross-linking activating and/or imaging moieties, or pharmaceuticallyacceptable salts thereof, as well as other moieties and/or agents and/orpharmaceutically acceptable salts thereof, are formulated as dry powdersor aerosolized physiologically acceptable solutions that may bedelivered to the lungs of a subject. Power and/or liquid formulationscan be prepared to facilitate administration, e.g., to facilitatetransfer from the delivery device into the respiratory tract, preferablydown to the alveoli distal to terminal bronchiles.

Powder formulations can be prepared in various ways, using conventionaltechniques. Powder formulations can be processed to improve ability tobe delivered to a subject, e.g., via inhalation and/ortrans-thoracically. For instance, the way in which the formulation flowsthrough and/or out of an inhaler device or other device, can be improvedby forming spherical agglomerates by, e.g., dry granulation processing.Spherical agglomerate can impart the glue compositions of this inventionwith superior handling characteristics. It is to be understood, however,that the present invention contemplates the use agglomerates and/orother particles of all shapes, including both spherical andnon-spherical shapes. Power and/or liquid formulations also preferablyhave physical characteristics that help avoid clogging of an aerosoldevice and clumping of aerosolized material. For example, additives suchas alcohol, soaps, surfactants, and/or Vitamin E may be use to helpreduce clumping and to facilitate formation of small particles and/ordroplets.

Liquid formulations may be produced by adding a volume of steriledelivery solvent to an amount of sterile composition of the presentinvention in powder or liquid form. In some embodiments, formulationtemperatures of at least about 0° C., at least about 4° C., at leastabout 5° C., at least about 10° C., or at least about 15° C. may beused. In some embodiments, formulation temperatures of less than about100° C., less than about 80° C., less than about 60° C., less than about37° C., or less than about 30° C. may be used.

Formulation of the present invention may also be prepared to provideother suitable physiological parameters for use in the lungs, includingfor example, suitable pH. For instance, a pH of at least about 4, atleast about 5, or at least about 6 may be used. In some embodiments, apH of less than about 11.0, less than about 10.0, less than about 9.0,less than about 8, or less than about 7 may be used.

In preferred embodiments, formulation involves selecting parameters suchas concentration, size and/or viscosity of adhering, cross-linkable,cross-linking activating and/or imaging moieties, as well as of othermoieties and/or agents, and/or pharmaceutically acceptable saltsthereof, e.g., to provide a rheological profile, such that whenaerosolized and/or nebulized, the formulation produces a range ofparticle and/or droplet sizes capable of being delivered to the lungs. Asuitable mill, such as a jet mill, can be used to produce particles in arange of sizes that facilitates, or preferably maximizes, access tosites of damaged lung tissue, including sites distal to terminalbronchioles. In some embodiments, a nozzle comprising tapering pores maybe used, e.g., to increase uniformity of the aerosol generated. See,e.g., U.S. Publication No. 2004/0124185.

In more preferred embodiments, a formulation is prepared that allowsrespiratory zone or deep lung delivery. In such embodiments, theformulation can yield a range of particle and/or droplet sizes adaptedfor delivery to the deep lung. In still more preferred embodiments,formulation involves selecting parameters such as concentration, sizeand/or viscosity of adhering, cross-linkable, cross-linking activatingand/or imaging moieties, as well as of other moieties and/or agentsand/or pharmaceutically acceptable salts thereof, such that whenaerosolized and/or nebulized, the formulation produces a range ofparticle and/or droplet sizes capable of being delivered to the lungalveoli, preferably to a lung alveolus distal to a terminal bronchiole,and more preferably to the deepest terminal branches of selected lungsegments.

Droplets and/or particles of suitable size ranges can be obtained byselecting appropriate delivery devices, molecular weight, concentration,and/or additives as known in the art and/or described herein. See, e.g.,U.S. Publication No. 2002/0086842. For example, various glueformulations can be screened to determine ones that produce dropletand/or particle size in desired ranges.

In preferred embodiments, the glue compositions of the present inventionare administered to a selected localized region of damaged lung tissuevia the respiratory tract, e.g., via inhalation. The term “inhalation”includes inhalation via the mouth, nose, tracheae, or any combinationthereof. A pharmaceutical formulation for administration via inhalationmay be made up according to techniques known in the pharmaceutical artsand administered via aerosol inhalation, dry powder inhalation, liquidinhalation, and/or instillation. For example, a diagnostically and/ortherapeutically effective amount of a glue composition of the inventionmay be delivered by inhalation of a breathable mist by the animalsubject.

Preparation of inhalable formulations are known in the art, e.g., seeU.S. Publication No. 2003/0232019 and International Publication No. WO2004/054556. For example, a glue composition of the present inventioncan be formulated with a breathable fluorocarbon propellant. Inhalablepreparations preferably provide droplets and/or particles with medianmass distribution size of at least about 0.1 microns, at least about 0.3microns, at least about 0.5 microns, at least about 1 micron, or atleast about 2 microns. Inhalable preparations preferably providedroplets and/or particles with median mass distribution size of lessthan about 20 microns, less than about 15 microns, less than about 10microns, less than about 6 microns, less than about 5 microns, less thanabout 3 microns, or less than about 2 microns. Particle and/or dropletsizes are preferably between about 2 microns to about 5 microns.

Size may be selected to allow glue compositions of the present inventionaccess to sites of damaged tissue in selected lung regions. Therespiratory system can be divided into three regions: (i) thetracheal/pharyngeal region, (ii) the bronchial region, and (iii) thealveolar region. Droplets and/or particles of about 10 microns to about50 microns typically migrate to the tracheal/pharyngeal and/or bronchialregion of the lungs; while droplets and/or particles of about 0.5microns to about 5 microns, e.g., droplets and/or particles of about 2microns, typically migrate to the alveolar region. Larger sizes may notas efficiently reach alveoli through distal bronchioles. Smallerdroplets and/or particles may be exhaled by the subject before theadhering moiety contacts and/or adheres to lung tissue. Droplet and/orparticle size of glue compositions of the present invention can bemeasured by techniques known in the art, including, e.g., thosedescribed herein.

Various physical parameters may be used to facilitate access of gluecompositions of the present invention to selected localized sites ofdamaged tissue within the lungs. For example, the mass medianaerodynamic diameter (MMAD), usually expressed in microns, can be usedto predict where a droplet and/or particle distributes in the lungs.Mass Median Aerodynamic Diameter can be measured using a CascadeImpactor relating to size of compositions of the present invention. Ahumidified Cascade Impactor is preferably used to better reflectconditions of pulmonary delivery. Further, particle size distributioncan also be measured with a Malvern Laser, for example. The geometricstandard deviation (GSD) is another parameter that can be used. A GSD ofabout 1 correlates to a normal distribution. A GSD of less than aboutone can indicate a narrow size dispersion while a GSD of more than about0.1 can indicate a broad size dispersion.

Charge may also be used to facilitate aerosol formation. For example, insome embodiments, droplets and/or particles can be made to carry anegative charge. The like charges can repel each other, helping todisperse the particles and/or droplets into an aerosol cloud by, e.g.,by electrostatic forces. Like positive charges on particles and/ordroplets may also be used in a similar manner.

Animal models can also be used to determine suitable ranges of dropletand/or particle size for delivery of glue compositions of the presentinvention to damaged lung tissue, e.g., see Raabe et al., Ann. Occup.Hyg., Vol. 32 pgs. 53-63 (1998) (surveying access of particle size tovarious regions of the lungs in laboratory animals).

Solution or liquid formulations may be aerosolized to form a breathablemist via, e.g., a device such as an inhaler, a nebulizer, and/or anatomizer. In some embodiments, the formulation is a dry power, which canbe made up into solution, e.g., with saline or water beforeaerosolization. In still some embodiments, a dry powder can be deliveredper se by a device such as an intra alveolar device (IAD), an air gunpowered aerosol chamber, and/or other dry powder delivery devices, e.g.,from Dura Delivery Systems and/or Glaxo Wellcome.

A glue composition of the present invention may be aerosolized by anytechniques known in the art, described herein and/or that can bedeveloped. For example, the glue composition may be pressurized throughmicro pores and then blown through an inline blower, such as ahigh-pressure fan system. The fan or pump is preferably timed tocoincide with the time of inspiration or a time just before inspiration.In some embodiments, for example, the delivery of the glue compositionscan be metered as a function of the in-flow volume.

The aerosolized composition can be delivered by any methods known in theart and/or described herein. For example, the glue composition can beinfused under pressure directly into a bronchus and/or into an enlargedair space. A catheter can be used to suck air out of a less distal lumenof the lungs through another path. In some embodiments, the gluecomposition can be infused into an enlarged air space using a firstcatheter while sucking air out with a second catheter through anotherpath leading from the same air space, e.g., from another bronchi branch,to get a circular flow path. In yet another approach, the flow around acatheter or other infusion device can be blocked using balloons, coveredbraid structures, expanding foam, flaps that make one-way valves, and/orexpanding corrugations.

Glue compositions of the present invention may also be administered viainhalation using a portable (e.g., hand held) inhaler device, such asdevices used to deliver anti-asthmatic agents or anti-inflammatoryagents. For example, a fine dry powder can be delivered as an aerosol bycompressing air into the powder inside the inhaler. This can dispersethe powder as a cloud of particles, preferably of the size ranges thatallow access to alveoli distal to terminal bronchioles.

In some embodiments, the inhaler device may be designed to deliversingle or multiple doses, minimizing risks from accidental large doses,and protecting the formulation from light, excessive moisture, and/orother contaminants. Dry powder and metered dose inhalers can be used toadminister glue compositions of the invention to the pulmonary airpassages of a subject in need thereof. Metered dose inhalers can delivermedicaments in a dispersion and/or in solubilized form. These inhalerscan include a relatively high vapor pressure propellant, which forcesaerosolized material into the respiratory tract upon activation of thedevice.

Some embodiments involve delivery by nebulization to the lungs, where,e.g., the delivery device can be a nebulizer. For example, a nebulizercan be used that generates an aerosol containing the glue compositionsof the present invention, preferably an aerosol of droplets and/orparticles of less than about 10 microns. Nebulizers are known in theart, and include, e.g., a jet nebulizer, which can be an air or liquidjet nebulizer; an ultrasonic nebulizer; a compressed air nebulizer(e.g., an AeroEclipse, Pari L. C., a Parijet; and/or a Whisper Jet)and/or a pressure mesh nebulizer. Compressed air nebulizers can generatedroplets by using fast moving air to shatter a liquid stream. Ultrasonicnebulizers can nebulize a liquid solution using ultrasonic waves, e.g.,by using a piezoelectric transducer to transform electrical current intomechanical oscillations; while pressure mesh nebulizers force fluidthrough a mesh-like surface under pressure. The nebulizer may use apressure of at least about 5 psi, at least about 10 psi, at least about15 psi, at least about 20 psi, at least about 25 psi, or at least about30 psi. The nebulizer may use a pressure of less than about 60 psi, lessthan about 50 psi, or less than about 40 psi. For administration using anebulizer, a subject can inhale aerosolized composition of the presentinvention via continuous neblulization, e.g., in a manner similar tothat used to administer aerosolized bronchodilators. For example, theaerosol may be delivered via tubing or a mask to the mouth and/or nose,as well as by using an Ambu bag, blow-by mask, endotracheal tube, nasalcannula, nasal covering, and/or nonrebreather.

A suitable volumetric flow rate (L/min) for the nebulizer may beselected. It is preferable that the volumetric flow rate not exceedtwice the subject's minute ventilation, as the average inspiratory rateis about twice the minute ventilation with exhalation and inhalationeach representing about half of the breathing cycle. For example, anebulizer with a volumetric flow rate of less than about 20 L/min, lessthan about 15 L/min or less than about 10 L/min may be used. A nebulizercan also be selected to generate desired ranges of particle and/ordroplet size. Along with volumetric flow rate, various factors may beconsidered as will be appreciated by one of skill in the art. Suchfactors include aerosol mass output (mg/L) and/or retained volume (mL).For example, with respect to a compressed air nebulizers, factors suchas air flow, hole diameter, and/or air pressure can influence sizedistribution. With respect to an ultrasonic nebulizer, factors includerate of air flow, hole diameter, and/or ultrasound frequency,

Administration can also involve delivery of aerosolized droplets and/orpowders of the present invention under positive pressure ventilation.For example, a device such as a Continuous Positive Airway Pressuredevice can be used to afford ventilatory assistance. This assistance canfacilitate access of the glue compositions of the present invention tosites of damaged tissue in alveoli of the deep airways. Additionally,positive end expiratory pressure may be used to provide furtherassistance in this regard. In some embodiments, a device can be usedthat delivers a glue composition of the present invention when thesubject produces a level of negative inspiratory pressure, e.g., atinspiratory flow rates.

Other devices that may be used include, for example, include a canisteradapted to contain a preparation comprising a glue composition of thepresent invention under pressure. The canister may feature a valve,e.g., for regulating delivery of the preparation; a nozzle connected tothe valve for converting the pressurized preparation inside the canisterinto an inhalable aerosol mist upon actuating the valve. See, e.g., U.S.Publication No. 2002/0086852. Other devices for delivery of gluecompositions of the present invention to the lungs of a subject in needthereof include a spray atomizer.

Glue compositions of the present invention can also be delivered in anon-aerosolized form. Further, any combination of aerosol and/ornon-aerosol forms may be used.

For example, a liquid, solution, suspension, viscous liquid, liquidfilm, slurry, foam, and/or thicksotropiec form(s) may be used. Any ofsuch forms can be delivered to selected localized regions of the lungsby any techniques known in the art, to be developed, and/or describedherein. For example, a liquid, solution, suspension, viscous liquid,liquid film, slurry, foam, and/or thicksotropiec form can beadministered by fluid washings, liquid ventilation, bolus liquid dripand/or pulmonary lavage, e.g., to a selected region of damaged lungtissue. In some embodiments, a fluorochemical medium may be used.

Administered solutions may include, for example, physiologicallyacceptable solutions of adhering, cross-linkable, cross-linkingactivating and/or imaging moieties (and/or other moieties and/or agents)of the present invention. After delivery at a selected region of damagedlung tissue, the solvent can evaporate and/or dissipate such that theadhering moiety, cross-linkable moiety, cross-linking activating and/orimaging moiety (and/or other moiety and/or agent) is left behind.

In still some embodiments; the glue compositions may be delivered assolids, semi-solids, solid films, hydrogels, agars, and/or sol-gels. Forexample, glue compositions of the present invention may be administeredto a selected localized region of the lungs as an absorbable sponge,e.g., as an absorbable gelatin sponge (e.g., GelfoaM™) and/or as anabsorbable wax. Non-absorbable waxes may also be used. Further, in someembodiments, petroleum-based compounds (e.g., petrolatum), latex,natural or synthetic rubber, starches, and/or alginate compounds may beused in formulating glue compositions of the present invention.

Aerosol and/or non-aerosol formulations can be delivered to a localizedregion of damaged lung tissue, preferably a localized region that hasbeen selected for volume reduction. For example, a localized region oflung tissue showing a large portion of damaged alveoli, e.g., “blebs” orother damage due to a pulmonary condition, such as emphysema, can beselected. The region identified via radiology for diagnosis, e.g., canbe located via bronchoscopy for treatment.

In some preferred embodiments, a bronchoscope is placed down the tracheaof a subject (e.g., an anesthetized intubated patient) and into abronchus, most preferably, as distally as possible to the selectedregion of damaged lung tissue. The anatomical site for administration,e.g., administration by a bronchoscope, may depend on the locationand/or extent of lung tissue damage. Within the respiratory tree,segmental bronchi branch and/or subdivide to produce subsegmentalbronchi, which in turn branch and/or subdivide to produce bronchiolesthat terminate in alveoli. The bronchoscope can be placed in the tracheaand advanced towards the branching bronchi. The bronchoscope that may beused is not limited, and may include, e.g., a rigid or fiberopticbronchoscope, e.g., a bronchoscope that allows visualization of anilluminated field. Use of an imaging moiety with a glue composition ofthe present invention can facilitate such visualization (e.g. usingfluoroscopy), as discussed in more detail below. Bronchoscopes that maybe used in the practice of the present invention include, for example,Fujinon, Olympus, and/or Pentax bronchoscopes.

A catheter can then be advanced through the bronchoscope, e.g., throughthe working channel of the bronchoscope. The catheter used is notlimiting, but preferably comprises a small diameter catheter, having adiameter at least less than that of the working channel of thebronchoscope. The catheter can be a single or dual lumen catheter. Thecatheter can be advanced beyond the trachea and bronchi, e.g., to reacha segmental bronchus, a subsegmental bronchus, a bronchiole and/or analveolus of the selected localized region of damaged lung tissue. Thecatheter can be allowed to become wedged in one or more of thesesegmental bronchi, subsegmental bronchi, bronchioles and/or alveoli,therein anchoring the distal tip of the catheter.

In some embodiments, the catheter used further comprises an expandablestructure at and/or near its distal tip, e.g., a balloon or balloon-likestructure. The expandable structure may be distended, e.g., with air,saline, any other suitable fluid and/or other medium, to assist, forexample, in anchoring the tip of the catheter and/or positioning it fordelivery of a glue composition of the present invention (in aerosoland/or non-aerosol form) to the selected localized region of damagedlung tissue. The balloon (or balloon-like structure) may be spherical,cylindrical, or any other shape. The distended balloon (or balloon-likestructure) may have a diameter of at least about 0.1 mm, at least about0.5 mm, at least about 1.0 mm, at least about 1.5 mm, at least about 3m, at least about 4 mm, at least about 5 mm, at least about 6 mm, atleast about 7 mm, at least about 8 mm, at least about 9 mm, or at leastabout 10 mm. The balloon diameter may be less than about 30 mm, lessthan about 20 mm, less than about 15 mm, or less than about 12 mm. Thediameter selected can help position the balloon (or balloon-likestructure) in a segmental bronchi, subsegmental bronchi, bronchioleand/or alveolus within a deep region of the lung. This anchoring and/orpositioning can facilitate delivery of the glue composition of thepresent invention to the selected localized region of damaged lungtissue. A glue composition may then be administered in aerosol and/ornon-aerosol forms, preferably at a controlled flow, to the selectedlocalized region of damaged lung tissue. The inflated balloon (orballoon-like structure) may additionally help contain the administeredglue composition to areas of the selected region of the lung that aredistal to the balloon (or balloon-like structure). That is, the balloon(or balloon-like structure) can help prevent the administered gluecomposition from spreading to other regions of the lungs other than theselected localized regions of damaged lung tissue.

A number of administrations may be carried out before removal of thebronchoscope. For example, compositions containing additional moieties,e.g., a cross-linking activating moiety, a sclerosing agent, and/or anyother moiety and/or agent, may also be administered at the same time orat separate times before removal. The balloon (or balloon-likestructure) may then be deflated and withdrawn through the bronchoscope.In some embodiments, additional glue composition is administered afterremoval of the catheter, e.g., to seal any gap due to the physicallocation of the balloon (or balloon-like structure). See, e.g.,Publication No. U.S. 2002/0147462. In some embodiments, glue compositionis delivered by the catheter distal to the balloon (or balloon-likestructure) so that no additional administration of glue composition isrequired.

As well as or instead of a catheter, forceps and/or other suitableinstruments may be used to deliver glue compositions of the presentinvention to a selected localized region of damaged lung tissue. Forexample, an endotracheal tube and/or applicator may be used and/or, insome embodiments, administration may achieved by laproscopy. In lesspreferred embodiments, administration can be achieved by open surgery,e.g., by a thracotomy, but less-invasive procedures are preferred, asindicated above.

In some embodiments, glue compositions of the present invention aredelivered to the lungs via instillation, e.g., direct instillationthrough the trachea, e.g., through the anterior aspect of the trachea.The glue compositions of the present invention can be administered as aliquid solution, including, e.g., an aqueous solution comprising wateror a buffered physiological solution, such as saline. Instillationadministration can be carried out over a period of at least about 2minutes, at least about 5 minutes, at or least about 10 minutes. Theinstillation period may be less than about 30 minutes, less than about20 minutes, or less than about 15 minutes. The length of instillationtime may be selected based on a number of factors, including the gluecomposition used, the size of the selected region of damaged lung tissueto be treated, the extent of the damage, and the like. Instillation mayinvolve delivery via bronchoscopy and/or endoscopy.

Other techniques for delivering glue compositions of the presentinvention to a selected localized region of damaged lung tissue may alsobe used, including, e.g., use of an impregnated applicator tip, e.g.,U.S. Pat. No. 5,928,611; and/or an applicator for delivering liquidand/or semi-liquid compositions via laproscopy and/or endoscopy e.g.,U.S. Pat. No. 6,494,896. Fibers, micro fibers, lattice-work stents,filagree designs, and/or porous structures may also be used, e.g., wherethe structure is coated with a glue composition of the present inventionand delivered to a selected localized region of damaged lung tissue

The glue compositions of the present invention can also be delivered viatrans-thoracic administration. For example, in some embodiments, airspaces of selected damaged regions of lung tissue can be targeteddirectly through the ribs for more controlled localization, e.g., beingapplied through a scope. Trans-thoracic delivery may involve deliveryinto the pleural space using a needle percutaneously and/or using acatheter and/or chest tube. Glue compositions of the present inventioncan also be delivered to the lungs during liquid ventilation orpulmonary lavage using a fluorochemical medium.

The glue compositions of the present invention can also be givenintravenously. For example, the pharmaceutical glue compositions of thepresent invention may be formulated with a pharmaceutically acceptablecarrier to provide sterile solutions or suspensions for administrationvia injection. Injectables can be prepared in conventional forms, e.g.,as liquid solutions, suspensions and/or solid forms suitable for makinga solution or suspension in liquid prior to injection, and/or asemulsions. Suitable excipients that may be used include, for example,water, saline, dextrose, mannitol, lactose, lecithin, albumin, sodiumglutamate, cysteine hydrochloride, and the like. In some embodiments,pharmaceutical compositions for injection may contain auxiliarysubstances, such as wetting agents, pH buffering agents, and the like.For example, a carbonate/bicarbonate buffer system may be used.

In some embodiments, the glue compositions of the invention areadministered using a delivery vehicle. A “delivery vehicle” as usedherein refers to any particle that can be used to carry compositions ofthe present invention. Examples of delivery vehicles include, but arenot limited to, liposomes, viral, bacteriophage, cosmid, plasmid, andfungal vectors and other recombinant vehicles typically used in the art.

Delivery vehicles can carry a glue composition of the present inventionencoded by a polynucleotide sequence. Expression of the sequence canproduce the glue composition, e.g., a fusion polypeptide of two or morecoupled adhering moieties. Vectors that contain both a promoter and acloning site into which a polynucleotide can be operatively linked arewell known in the art. Such vectors are capable of transcribing RNA invitro or in vivo, and are commercially available from sources such asStratagene (La Jolla, Calif.) and Promega Biotech (Madison, Wis.). Inorder to enhance in vitro transcription and/or expression, it may benecessary to remove, add, and/or alter 5′ and/or 3′ untranslatedportions to eliminate extra, potentially inappropriate alternativetranslation initiation codons, or other sequences that may interferewith or reduce expression, either at the level of transcription ortranslation. In some embodiments, consensus ribosome binding sites canbe inserted immediately 5′ of the start codon to enhance expression.

In some embodiments, a viral vector can be used. A viral vector caninclude a natural or recombinantly produced virus or viral particle thatcomprises a polynucleotide to be delivered, either in vivo, ex vivo orin vitro. Examples of viral vectors include baculovirus vectors,retroviral vectors, adenovirus vectors, adeno-associated virus vectorsand the like. A viral vector can enter a host cell via its normalmechanism of infection or can be modified such that it binds to adifferent host cell, e.g., by binding to a different surface receptor orligand to enter the different host cell.

Delivery vehicles can also include non-viral vectors, including liposomecomplexes. Liposomes may comprise an aqueous concentric layer adherentto a hydrophopic or lipidic layer. The hydrophobic layer may comprise,for example, phospholipids, such as lecithin and sphingomyelin, steroidssuch as cholesterol, as well as ionic surface active substances such asdicetyphosphate, phosphatidic acid, stearylamine, and the like. Variousliposome complexes known in the art may be used to aid delivery of theglue compositions of the invention to the lungs, in aerosol and/ornon-aerosol formulation. For example, particulate formulations combiningcompounds having biocompatible hydrophobic domains with conjugateshaving both hydrophobic and hydrophilic regions may be used. See, e.g.,U.S. Pat. No. 6,500,461. In some embodiments, lipid vesicles may be usedcomprising bilayers with a salt form of an organic acid derivative of asterol, as described, e.g., in U.S. Pat. No. 6,352,716. In someembodiments, the use of liposome complexes can facilitate delivery ofglue compositions of the present invention, e.g., by keeping the gluecomposition intact and/or in appropriate conformation necessary and/orinvolved in adhering to lung tissue.

In still some embodiments, liposomes containing compositions of theinvention are coated with, e.g., a hydrophilic agent, such ashydrophilic polymer chains like polyethylene glycol (PEG). Examples ofPEG-liposomes are known in the art, e.g., see U.S. Publication No.2003/0138481 and U.S. Publication No. 2003/0113369. In some embodiments,the adhering moiety may be coupled to exposed PEG chains to facilitateadhering to lung tissue. In some embodiments, the hydrophilic chains maytemporarily shield the adhering moiety from interaction with lungtissue. Such liposomes are described, e.g., in U.S. Publication No.2004/0009217.

In some embodiments, liposome complexes may facilitate selectivedelivery to localized regions of damaged lung tissue. For instance,peptide-lipid conjugates may be incorporated into liposomes, for exampleto selectively destabilize the liposomes in the vicinity of high amountsof elastase or other peptidases, as found in certain pulmonaryconditions. See, e.g., peptide-lipid conjugates described in U.S. Pat.No. 6,087,325.

Delivery vehicles can also include other delivery systems associatedwith membranes (e.g., biocompatible or bioerodable membranes),including, e.g., dendrimer-based methods. See, e.g., US Publication No.2004/0120979. See also, e.g., US Publication No. US2003/0064050,describing dendritic polymer conjugates useful as drug delivery systems.For example, a dentritic polymer conjugate useful as a delivery systemin the practice of the present invention can comprise a dendriticpolymer coupled to an adhering moiety described herein.

In some embodiments, a glue composition of the present invention may beused with a moiety that increases solubility and/or pharmacologiccompatibility of the adhering, cross-linkable, cross-linking activatingand/or imaging moiety, as well as other moieties and/or agents, forexample, by enhancing hydrophobicity. For example, in some embodiments,absorption enhancing preparations (e.g., liposomes described above) maybe utilized. Moieties that may be co-administered to achieve sucheffects include, for example, amphotericin B, betamethasone valerete,beclomethasone, cortisone, dexamethasone, DPPC/DPPG phospholipids,doxorubicin, estradiol, isosorbide dinitrate, nitroglycerin,prostaglandins, progesterone, testosterone, and/or vitamin E, and/oresters of any of these.

Glue compositions for use in treating pulmonary conditions preferablyhave low levels of toxicity during useable life and are preferablysterilized. Sterilization may be accomplished by techniques known to inthe art, including, for example, chemical, physical, and/or irradiationmethods. Physical methods can include sterile fill, filtration, use ofheat (dry or moist) and/or retort canning. Irradiation methods ofsterilization can include gamma irradiation, electron beam irradiation,and/or microwave irradiation. Preferred methods are dry and moist heatsterilization and electron beam irradiation. Different moieties of theinvention can be sterilized separately, e.g., as described in EP1433486, e.g., to form final sterile glue compositions.

Preferably, the glue compositions of the present invention have abacterial count of less than about 2 cfu/g, less than about 1 cfu/g, orless than about 0.1 cfu/g. Such precautions can reduce abscessformation. Preservatives may also be used including, but not limited to,hydroquinone, pyrocatechol, resorcinol, 4-n-hexyl resoreinol, captan(i.e.,3α,4,7,7α-tetrahydro-2-((trichloromethyl)thio)-1H-isoindole-1,3(2H)-dione),benzalkonium chloride, benzalkonium chloride solution, benzethoniumchloride, benzoic acid, benzyl alcohol, cetylpyridinium chloride,chlorobutanol, dehydroacetic acid, o-phenylphenol, phenol, phenylethylalcohol, potassium benzoate, potassium sorbate, sodium benzoate, sodiumdehydroacetate, sodium propionate, sorbic acid, thimerosal, thymol,phenylmercuric compounds such as phenylmercuric borate, phenylmercurienitrate and phenylmercuric acetate, formaldehyde, and formaldehydegenerators such as the preservatives Germall II® and Germall 115™(imidazolidinyl urea, available from Sutton Laboratories, Charthan,N.J.), and the like. Further, preferred preparations contain nontoxicconcentrations of toxins, such a heavy metals, for example, usingestablished criteria for USP water for inhalation.

The present invention also encompasses pharmaceutical glue compositionsprepared for storage before administration. Such compositions preferablycontain preservatives and/or stabilizers. For example, sorbic acidand/or esters of phydroxybenzoic acid may be added. In addition,antioxidants and suspending agents may be used.

Pharmaceutical glue compositions useful in this invention may alsoinclude stabilizing agents, e.g., to reduce premature cross-linking.Stabilizing agents can include, e.g., vapor phase stabilizers, such asan anionic vapor phase stabilizer, and/or liquid phase stabilizers,e.g., an anionic liquid phase stabilizer. Such stabilizing agents mayalso include radical stabilizing agents, and/or a mixture of variousstabilizing agents, preferably where the mixture does not interferewith, retard, and/or prevent the desired reaction. See, e.g., U.S.application Ser. No. 09/099,457.

If necessary or desirable, the glue compositions of the presentinvention may be administered in combination with one or more othertherapeutic agents. The choice of therapeutic agent that can beco-administered with a glue composition of the present invention willdepend, in part, on the condition being treated and the desired effectto be achieved.

For example, the glue composition may be administered with a growthfactor, an anti-surfactant and/or an antibiotic or other therapeuticagent, including small molecule or polypeptide drugs. Examples of growthfactors that may be used include a fibroblast growth factor, atransforming growth factor-β₁, and/or a platelet-derived growth factor(PDGF), as well as functional analogs thereof. Determination of dosageranges are well within the knowledge and/or skill of those in the art,e.g., about 1 to about 100 nM of polypeptide growth factor can be used.

Examples of antibiotics that may be used include ampicillin, sisomicin,cefotaxim, gentamycin, penicillin, nebacetin, and the like.Additionally, in some embodiments, antimicrobial agents, antiviralagents, antiseptics, bacteriocins, disinfectants, anesthetics,fungicides, anti-inflammatory agents, or other active agents or mixturesthereof may be administered with a glue composition of the presentinvention. Such compounds can include acetic acid, aluminum acetate,bacitracin, bacitracin zinc, benzalkonium chloride, benzethoniumchloride, betadine, captan (i.e.,3α,4,7,7α-tetrahydro-2-((trichloromethyl)thio)-1H-isoindole-1,3(2H)-dione),benzalkonium chloride, benzalkonium chloride solution, benzethoniumchloride, benzoic acid, benzyl alcohol, bleomycin, calciumchloroplatinate, cephalosporin, certrimide, cetylpyridinium chloride,chlorobutanol, cloramine T, chlorhexidine phosphanilate, chlorhexidine,chlorhexidine sulfate, chloropenidine, chloroplatinatic acid,ciprofloxacin, clindamycin, clioquinol, cresol, chlorocresol,cysostaphin, dehydroacetic acid, doxorubicin, formaldehyde, gentamycin,hydroquinone, hydrogen peroxide, iodinated polyvinylidone, iodine,iodophor, imidazolidinyl urea, minocycline, mupirocin, neomycin,neomycin sulfate, nitrofurazone, non-onynol 9, o-phenylphenol,phenylmercuric additives such as phenylmercuric borate, phenylmercurienitrate and/or phenylmercuric acetate phenol, phenylethyl alcohol,potassium benzoate, potassium sorbate, potassium permanganate,polymycin, polymycin B, polymyxin, polymyxin B sulfate,polyvinylpyrrolidone iodine, povidone iodine, 8-hydroxyquinoline,preservatives (e.g., alkyl parabens and salts thereof, such asbutylparaben, ethylparaben, methylparaben, methylparaben sodium,propylparaben, propylparaben sodium, and/or pyrocatechol), quinolonethioureas, rifampin, rifamycin, resorcinol, 4-n-hexyl resoreinol, silveracetate, silver benzoate, silver carbonate, silver chloride, silvercitrate, silver iodide, silver nitrate, silver oxide, silver sulfate,sodium benzoate, sodium dehydroacetate, sodium propionate, sorbic acid,sodium chloroplatinate, sodium hypochlorite, sphingolipids, sulfonamide,tetracycline, sulfadiazine salts (such as silver, sodium, and zinc),thimerosal, thymol, tiotropium bromide, zinc oxide, and the like, andany combinations thereof.

Other drug moieties that may be co-administered include, for exampleanti-oxidants, atropine methyl nitrate, albuterol (salbutamol) sulfate,alcetylcysteine, anticholinergics, atriopeptin, bitolterol mesylate,beta agonists, other bronchodilators, e.g., isoetharine,methylxanthines, captopril, calcitonin, cromolyn sodium, cyclosporin,ephedrine sulfate, ephedrine bitartrate, epidermal growth factor,etoposide, fluoroisolide, heparin, ibuprofin, insulin, interferon,isoetharine hydrochloride, insulin, interleukin-2, isoetharine mesylate,isoproteranol hydrochloride, isoproteranol sulfate, leukotrieneinhibitors, lipase inhibitors, lipocortin, lung surfactant protein, mastcell stabilizers, metaproteranol sulfate, narcotics, n-acetyl cysteine,pentamidin, non-steroidal anti-inflammatory drugs (NSAIDs), peptides,phosphodiesterase inhibitors, phospholipase inhibitors, plasma factor 8,procaterol, propranalol, pulmozyme (Genentech), P2Y2 receptor agonists,steroids, superoxide dismutase, terbutaline, terbutaline sulfate,theophylline, tissue plasminogen activator (TPA), tobermycin, tumornecrosis factor, vasopressin, and/or verapamil.

Further, the glue composition may also be administered with a nucleicacid, e.g., a nucleic acid encoding a polypeptide, antisenseoligonucleotide, or interfering RNA (e.g., siRNA). Compositions of thepresent invention may also serve as “depot” for slow release oftherapeutic moieties or other active agents at selected localizedregions of damaged lung tissue.

All formulations for aerosol, trans-thoracic, instillation, intravenousand/or other administration can be formulated in dosages suitable foradministration. Pharmaceutical compositions suitable for use in thepresent invention include glue compositions wherein the moieties and/oragents are present in an effective amount, i.e., in a diagnosticallyand/or pharmaceutically effective amount. A diagnostically effectiveamount includes a sufficient amount of a glue composition comprising animaging moiety to allow detection of the presence of the imaging moiety,preferably at a site of sealed lung tissue, and more preferably by anon-invasive and/or in vivo imaging technique. A pharmaceuticallyeffective amount includes a sufficient amount of a glue compositioncomprising an adhering moiety, cross-linkable moiety, cross-linkingactivating moiety and/or other agent to produce a therapeutic and/or aprophylactic benefit in at least one pulmonary condition being treated.The effective amount can be administered in a single dose or in a seriesof doses separated by appropriate time intervals, such as minutes, hoursor days. The actual amount effective for a particular application willdepend on the pulmonary condition being treated, the route ofadministration used, the identity of the adhering, cross-linkable,cross-linking activating and/or other moieties and/or agents to be used,and other considerations that will be appreciated by those of skill inthe art. Determination of an effective amount is well within thecapabilities of those skilled in the art, especially in light of thedisclosures herein.

The effective amount when referring to a glue composition comprising anadhering, cross-linkable, cross-linking activating, imaging moietyand/or other moiety and/or agent will generally mean the dose ranges,modes of administration, formulations, etc., that have been recommendedor approved by any of the various regulatory or advisory organizationsin the medical or pharmaceutical arts (e.g., FDA, AMA) or by themanufacturer or supplier. The effective amount when referring toproducing a benefit in treating a pulmonary condition, such asemphysema, will generally mean the amount that achieves clinical lungvolume reduction recommended or approved by any of the variousregulatory or advisory organizations in the medical or surgical arts(e.g., FDA, AMA) or by the manufacturer or supplier.

A person of ordinary skill using techniques known in the art candetermine the effective amount of the adhering moiety, cross-linkablemoiety, cross-linking activating moiety and/or other moiety and/or agentof the glue composition to be administered. The effective amount maydepend on the moiety and/or agent to be used, and can be deduced fromknown data, e.g., data regarding binding constants for an adheringmoiety, concentrations to achieve cross-linking for cross-linkable andcross-linking activating moieties, and sufficient imaging moiety topermit detection.

In some embodiments, dosages can be at least about 0.001 μg/kg/bodyweight, at least about 0.005 μg/kg/body weight, at least about 0.01μg/kg/body weight, at least about 0.05 μg/kg/body weight, or at leastabout 0.1 μg/kg/body weight. In some embodiment, dosages can be lessthan about 0.05 mg/kg/body weight, less than about 0.1 mg/kg/bodyweight, less than about 0.5 mg/kg/body weight, less than about 1mg/kg/body weight, less than about 2 mg/kg/body weight, less than about3 mg/kg/body weight, or less than about 5 mg/kg/body weight of acomposition of the invention. In some embodiment, dosages can be lessthan about 10 mg/kg/body weight, less than about 25 mg/kg/body weight,less than about 50 mg/kg/body weight, less than about 75 mg/kg/bodyweight, less than about 100 mg/kg/body weight, less than about 150mg/kg/body weight, or less than about 200 mg/kg/body weight of acomposition of the present invention.

The dosage may vary depending on the moieties used and their knownbiological properties. For example, it is known that fibrinogencomprises about 2 to about 4 g/L blood plasma protein and is cleaved tofibrin upon exposure to thrombin at the initiation the blood clottingcascade. In the context of reducing lung volume, formulations can beprepared containing useful concentrations of fribnogen and/or fibrin asa cross-linkable moiety and thrombin, batroxobin, a thrombin receptoragonist, and/or calcium as a cross-linking activating moiety. Forexample, a formulation comprising at least about 1%, at least about 2%,at least about 3%, at least about 4%, at least about 5%, at least about8%, at least about 10%, at least about 12%, or at least about 15%fibrinogen may be used (e.g., in saline solution, for instance about0.8%, about 0.9%, about 1%, or about 1.2% saline), and may be activatedusing at least about 0.5, at least about 1, at least about 5, at leastabout 10, or at least about 12 units of thrombin per ng of fibrinogen,and/or more than about 1 mM, more than about 1.5 mM, more than about 3mM, more than about 5 mM, or more than about 8 mM calcium (e.g., in aCaCl₂ solution). Some embodiments may use a preparation of less thanabout 40 mM, less than about 30 mM, or less than about 20 mM calcium(e.g., in a CaCl₂ solution). Additionally, at least about 0.5%, at leastabout 1%, at least about 3%, at least about 5%, or at least about 6% offactor XIIa transglutaminanse may also be used to promote cross-linking.Formulation of fibrin-based compositions for achieving cross-linking arealso known in the art, e.g., and may contain about more than about 10mg/ml, more than about 20 mg/ml, more than about 25 mg/ml, or more thanabout 50 mg/ml. Fibrin-based compositions useful in the practice of thisinvention may also contain less than about 250 mg/ml, less than about200 mg/ml, less than about 150 mg/ml, less than about 100 mg/ml, or lessthan about 50 mg/ml. See, e.g., other fibrin sealant compositions asprovided in e.g., U.S. Pat. No. 5,739,288.

Further, the effective amount for use in humans can be determined fromanimal models, e.g., mice, rabbits, dogs, sheep, or pigs. For example,emphysema can be induced in C57BL/6 mice by administering nebulizedporcine pancreatice elastase (about 30 IU/day for about 6 days), asdescribed, for instance, in Ingenito et al., Tissue heterogeneity in themouse lung: effects of elastase treatment, Articles in Press. J ApplPhysiol (Mar. 12, 2004). 10.1152/japplphysiol.01246.2003. Similarly,emphysema-like conditions may be induced in sheep exposed to papain(inhalation of about 7,000 units/week for four consecutive weeks).Emphysema can also be induced in animal models by exposure to cadmiumchloride, high concentrations of oxygen, and/or cigarette smoke.Ingenito, et al., “Bronchoscopic lung volume reduction using tissueengineering principles”, American Journal of Respiratory and CriticalCare Medicine, Vol. 167 pgs. 771-778 (2003). A dose suitable for sealingdamaged lung tissue in humans can be formulated based on doses found tobe effective in animal models in reducing lung volume and freeing upspace for expansion of remaining non-damaged or healthier tissue. Othertechniques would be apparent to one of ordinary skill in the art.Further, the amount of administered glue composition comprising across-linkable moiety and/or coupled adhering moieties can be selectedto be not so large as to generate high local hydrostatic pressures,preferably avoiding local hydrostatic pressures that exceed capillaryperfusion pressure that can lead to abscess formation.

Similarly, a dose for imaging damaged lung tissue in humans can beformulated based on that used to image the lungs of a suitable animalmodel. Diagnostic compositions comprising an adhering moiety and animaging moiety can be prepared using a pharmaceutically acceptablecarrier and a diagnostically effective amount of the glue composition.Diagnostically effective amount required as a dose to allow imaging willdepend upon the route of administration, the condition being treated,the adhering moiety being used, the imaging moiety being used, theimaging detail sought to be obtained, e.g. the extent of sealingachieved, as well as other factors that will be appreciated by those ofskill in the art of medical diagnostics. One of skill in the art ofmedical diagnostics will readily be able to determine suitable dosages,especially in light of the disclosures provided herein.

In preferred embodiments, the dose for imaging is sufficient to detectthe presence of an imaging moiety at a site of damaged (preferablysealed) lung tissue. For example, in some embodiments, radiologicalimaging can require that the dose provide at least about 3 μC, at leastabout 5 μC, or at least about 10 μC of imaging moiety. In someembodiments, radiological imaging can require that the dose provide lessthan about 30 less than about 20 μC, or less than about 15 μC of imagingmoiety. Some embodiments using magnetic resonance imaging can require adose of at least about 0.0005 mmol/kg, at least about 0.001 mmol/kg, atleast about 0.005 mmol/kg, at least about 0.01 mmol/kg, at least about0.05 mmol/kg, at least about 0.1 mmol/kg, at least about 0.5 mmol/kg, orat least about 1 mmol/kg of imaging moiety to body weight of thesubject. In some embodiments, magnetic imaging can require a dose ofless than about 10 mmol/kg, less than about 8 mmol/kg, less than about 5mmol/kg, less than about 3 mmol/kg, or less than about 2 mmol/kg of animaging moiety to the body weight of the subject. As a further example,iodine may be used as an imaging moiety in a dose of at least about 2mol percent, at least about 5 mol percent, at least about 7 mol percent,or at least about 8 mol percent of the administered glue composition.The iodine imaging moiety can be in a dose of less than about 20 molpercent, less than about 15 mol percent, less than about 12, or lessthan about 10 mole percent of the administered glue composition.

The exact dosage will be determined by the practitioner, in light offactors related to the subject in need of diagnosis and/or treatment.Factors which may be taken into account include the severity or extentof the pulmonary condition, the general health of the subject, age,weight, and diet of the subject, as well as the timing and frequency ofadministration, other diagnostic and/or therapeutic techniques availableand/or desirable to the subject, and/or being used by the subject, aswell as reaction sensitivities, allergies, tolerance and/or response tothe glue composition(s) of the present invention.

Methods of Treating Pulmonary Conditions

The present invention provides methods of treating pulmonary conditionsusing compositions that adhere lung tissue, including damaged lungtissue. The term “pulmonary condition” as used herein refers to anon-normal condition of the lungs and/or lung tissue, for example, wherethere is damaged lung tissue. Examples of such pulmonary conditionsinclude COPD, which includes emphysema, (including both heterogeneousemphysema and homogenous emphysema, preferably heterogeneous emphysema),asthma, bronchiectais, and chronic bronchitis. Pulmonary conditions canalso include other chronic pulmonary disorders, sarcoidosis, pulmonaryfibrosis, pneumothorax, fistulae, bronchopleural fistulae, cysticfibrosis, inflammatory states, and/or other respiratory disorders.Pulmonary conditions can also include smoking-related and/or age-relatedchanges to the lung, as well as lung damage caused by a traumatic event,infectious agents (e.g., bacterial, viral, fungal, tuberculin and/orviral agents), exposure to toxins (e.g., chemotherapeutic agents,environmental pollutants, exhaust fumes, and/or insecticides), and/orgenetic factors (e.g., alpha-1 antitrypsin deficiency and other types ofgenetic disorders which involve elastic and/or connective tissuedegradation and/or impaired synthesis of elastic and/or connectivetissues and/or impaired repair of elastic and/or connective tissues ofthe lungs).

One aspect of the present invention provides a method of reducing lungvolume by providing a glue composition comprising a cross-linkablemoiety and an adhering moiety wherein said moieties are coupled andwherein said adhering moiety adheres lung tissue; administering saidglue composition to a localized region of damaged lung tissue of asubject; collapsing a first portion or all of the lung of said subjectwherein said first portion comprises said localized region of damagedlung tissue; cross-linking damaged lung tissue; and re-inflating asecond portion of the lung of said subject wherein said second portiondoes not comprise said damaged lung tissue, thereby reducing lungvolume.

In some preferred embodiments, the method is performed with prioridentification of the damaged lung tissue. For example, the lungs of thesubject may be imaged to identify regions or sites of damaged tissuebefore administering a glue composition of the invention to the subject,e.g., to determine regions that may benefit from volume reduction. Asused herein, the terms “regions,” “sites,” and “areas” are usedinterchangeably when referring to regions, sites and/or areas of damagedlung tissue, e.g., localize regions, sites and/or areas of damaged lungtissue selected for administration of a glue composition of the presentinvention. Such identification may involve any techniques known, to bedeveloped, described herein, and/or described in U.S. nonprovisionalapplications entitled “Targeting Damaged Lung Tissue UsingCompositions,” filed Dec. 8, 2004; “Targeting Damaged Lung Tissue,”filed Dec. 8, 2004; “Targeting Sites of Damaged Lung Tissue UsingComposition,” filed Dec. 8, 2004; “Targeting Sites of Damaged LungTissue,” filed Dec. 8, 2004; “Imaging Damaged Lung Tissue UsingCompositions,” filed Dec. 8, 2004; “Imaging Damaged Lung Tissue,” filedDec. 8, 2004; “Glue Compositions for Lung Volume Reduction,” filed Dec.8, 2004; “Lung Volume Reduction Using Glue Compositions,” filed Dec. 8,2004; and “Lung Volume Reduction Using Glue Composition,” filed Dec. 8,2004, each of which is herein incorporated in its entirely thatfacilitate identification of regions of damaged lung tissue. Regions ofdamaged lung tissue include areas of the lung affected by a pulmonarycondition or that are affected to a greater extent compared with other,healthier areas of the lung. In emphysema, for example, such regions caninclude regions featuring “blebs,” that is, regions where progressivedestruction of elastic tissue has caused loss of lung recoil andconsequent hyper-extension.

Current techniques that may be used to identify damaged lung tissue inthe present invention include radiology (e.g., chest X-rays) and CTscans. For example, review of CT scans of the chest, preferablyhigh-resolution CT scans, can indicate localized regions of damaged lungtissue that may be selected for volume reduction.

Cross-linking of the damaged lung tissue can then bring about areduction in lung volume, for example, by sealing and/or keepingcollapsed regions of over-inflated lung tissue, preferably freeing upspace for the expansion of remaining non-damaged or healthier tissue. Inemphysema, for instance, regions of the lung that have lost elasticityrequired for exhalation can be collapsed and/or sealed by the methodsdescribed herein. Because the cross-linked tissue occupies a smallervolume than, e.g., the enlarged alveoli at sites of damaged tissue,methods of this invention can reduce lung volume overall. The presentinvention can thus provide a non-surgical, less-invasive and/or saferapproach for achieving some of the benefits of lung volume reductionsurgery. Further, providing the glue composition to a localized regionof damaged lung tissue allows for localized volume reduction, which inturn can minimize untoward side effects of lung volume reduction, suchas exacerbating V/Q imbalance, changing arterial oxygenation, ortriggering acute hypoxemia. Ingenito et al., (2002) Bronchoscopic LungVolume Reduction Using tissue engineering principles, American Journalof Respiratory and Critical Care Medicine, Vol. 167 pgs 771-778. It isto be understood also that the methods of the present invention may beused in conjunction with a surgical procedure, such as LVRS and the useof knifeless staplers (see, e.g., Swanson et al., “No-cut thoracoscopiclung plication: A new technique for lung volume reduction surgery”, J AmColl Surg Vol. 185 pgs 25-32 (1997)), as well as other approaches fortreating pulmonary conditions, including use of coupled adheringmoieties described herein.

Cross-linking of the cross-linkable moieties can be achieved by anymethods known in the art and/or described herein. For example, a secondcomposition may be administered that comprises a cross-linkingactivating moiety. “Cross-linking activating moiety” as used hereinrefers to any moiety that can bring about cross-linking between morethan one cross-linkable moieties and/or that can form more than one bondwith components (e.g., proteins) of lung tissue. Preferably, across-linking activating moiety comprises a di- or polyfunctional group.For example, where the cross-linkable moiety is at least one of ahydroxyl group, a carboxyl group, an ester group, a cyano group, a thiolgroup (e.g., a cysteine group), a carbonyl group, an aldehyde group, aketone group, a primary amine group, a secondary amine group, and/or alysine group the cross-linking activating moiety may comprise a diol, apolyol, a dicarboxylic acid (e.g., fumaric, maleic, phthalic orterephthalic acid), a polycarboxylic acid, a diester, a polyester, adiamine and/or a polyamine. The di- or polyfunctional group can formcovalent linkages with more than one cross-linkable moieties, preferablybetween cross-linkable moieties coupled to adhering moieties binding todifferent sites of damaged lung tissue, e.g., at different sites withinan enlarged alveolus. Linkage may include, for example, amide formation(e.g., through the condensation of an amino group with an activatedester, such as, e.g., an NHS or sulfo-NHS ester), imine formation,carbodiimide condensation, disulfide bond formation, and/or use of aspecific binding pair e.g., using a biotin-avidin interaction. Thecross-linking can therefore serve to seal and/or keep collapsed airspaces at sites of damaged lung tissue, e.g., in areas of over-inflatedalveoli, as characteristic of certain pulmonary conditions, includingemphysema.

Di- and/or polyamines that may be used in the practice of this inventioninclude aliphatic and/or aromatic di- and/or polyamines, as well as twoor more aliphatic and/or aromatic monoamines suitably linked together.For example, monomeric, di- and/or polyamines that may be used in thepractice of this invention can comprise aminopyrimidine, aniline,benzidine, diaminodiphenylamine, diphenylamine, hydrazine, hydrazide,toluene-diamine, and/or triethylenediamine. Di- and/or polyamines thatmay be used also can comprise, for example, acetamide, acrylamide,benzamide, cyanamide, and/or urea. Di- and/or polyalcohols that may beused include aromatic and/or aliphatic alcohols, including, for example,1,4-butanediol, phenols, polyvinyl alcohols, and/or d-sorbitol. Examplesof dicarbonyls that may be used in the practice of the present inventioninclude dicarbonyls comprising acetate, e.g., α-haloacetate derivatives,acetylacetone, diethylmalonate, ethylacetone, malonamide, malonic acidand/or malonic esters or salts thereof. Other carbonyl groups that maybe used include α, β-unsaturated carbonyl groups (e.g., maleimide)and/or α-halocarbonyl groups (e.g., iodoacetamide derivatives). Di-and/or polyfunctional ketones may also be used, including, e.g.,2,5-hexanedione, and/or di- and/or polyfunctional ketones comprising twoor more linked monofunctional ketones, such as cyclohexanone and/orcyclopentanone. Di- and/or polyfunctional aldehydes may also be used,see, e.g., U.S. Pat. No. 6,329,337 and/or U.S. Pat. No. 6,372,229. Forexample, at least one aldehyde selected from gelatin-resorcin-aldehyde,glyoxal, succinaldehyde, glutaraldehyde, malealdehyde,dextrandialdehyde, and saccharides oxidized by m-periodate may be used.

As will be appreciated by one skilled in the art, aldehydes and/orketones described herein can exist as hydrates in aqueous solution,e.g., existing as hemi-acetals and/or hemi-ketals in aqueous solution.In preferred embodiments, such hydrates can revert back to thecorresponding aldehyde and/or ketone for cross-linking. In someembodiments, hydrates of aldehydes and/or ketones and/or hydrates ofother cross-linking activating moieties are themselves capable ofbringing about cross-linking between more than one cross-linkablemoieties and/or of forming more than one bond with components (e.g.proteins) of lung tissue.

Other cross-linking activating moieties that may (or may not) be used inthe practice of the present invention include a protein or a mixture ofproteins (including synthetic peptides and/or recombinant proteins),such as collagen and/or albumin and/or lipoprotein, along with otherminor additives, optionally as well as hydrogel, polyglycolic acid,polylactic acid, polydioxanone, polytrimethylene carbonate,polycarprolactone, and/or glutaraldehyde, polyethylene glycol,polyethlyene glycol disuccinimidoyl succinate, as well as polymerizablemonomers, such as 1,1-disubstituted ethylene monomers or acetates, e.g.,a-haloacetate, acrylate, acrylate glue, anhydrides cross-linked withpolyols, cyano groups, e.g., cyanoacrylate, epoxy, gelatin resorcinolformaldehyde, gelatin resorcinol glutaraldehyde, hyaluronic acidcross-linked with hydrazines, photopolymerizable monomers, silicone,silicone rubber, starches, urethane, vinyl-terminated monomers, and/orany combination thereof. Other cross-linking activating moieties thatmay be used in the practice of the present invention include alkylbis(2-cyanoacrylate), triallyl isocyanurate, alkylene diacrylate,alkylene dimethacrylate, and/or trimethylol propane triacrylate. Othercross-linking activating moieties that may be used in the practice ofthe present invention include disulfide, carbodiimide and hydrazine.Other suitable cross-linking activating moieties may be found in theart, for example, U.S. Pat. No. 3,940,362; U.S. Pat. No. 5,328,687; U.S.Pat. No. 3,527,841; U.S. Pat. No. 3,722,599; U.S. Pat. No. 3,995,641;and/or U.S. Pat. No. 5,583,114, each incorporated herein by reference.Still another cross-linking activating moiety that may be used includesa product formed by reacting glutaraldehyde with amino acids and/orpeptides, as described in U.S. Pat. No. 6,310,036. Cross-linkable and/orcross-linking activating moieties may also include suitable monomersdisclosed in U.S. Publication No. 2002/0147462, such as, for instance,monomeric n-butyl-2-cyanoacrylate (Eng et al., “Successful closure ofbronchopleural fistula with adhesive tissue”, Scand J Thor CardiovascSurg, Vol. 24 pgs 157-59 (1990) and Inaspettato et al., “Endoscopictreatment of bronchopleural fistulas using n-butyl-2-cyanoacrylate”,Surgical Laparoscopy & Endoscopy, Vol. 4 No. 1 pgs 62-64 (1994)).

The choice of cross-linking activating moiety can depend, at least inpart, on the cross-linkable moieties used. Where the cross-linkablemoiety is fibrin and/or fibrinogen, the cross-linking activating moietymay comprise a fibrin activator and/or a fibrinogen activator. Forexample, thrombin, a thrombin receptor agonist, batroxobin, and/orcalcium can be used to initiate cross-linking of fibrinogen. It is alsoto be understood that any combination of cross-linking activatingmoieties may be used, depending on, for example, the combination ofcross-linkable moieties administered. Further, some embodiments providea glue composition comprising an adhering moiety coupled to across-linking activating moiety. Those of skill in the art willrecognize other suitable cross-linking activating moieties that may beused in the practice of the instant invention, including, for example,any biocompatible cross-linking activating moiety that can form abiocompatible cross-linked product with a cross-linkable moiety used. Instill more preferred embodiments, the cross-linkable and cross-linkingactivating moieties used are medically acceptable and form medicallyacceptable cross-links.

In some embodiments, one or more of the cross-linkable, adhering and/orcross-linking activating moieties are thermally stabilized. That is, themoiety may be modified, adapted and/or otherwise engineered to withstandheat, e.g., heat generated by a cross-linking reaction within lungtissue of a subject. For example, heat-stabilized glutaraldehyde in anaqueous carrier may be used, and in some embodiments amino acidmodifications in protein adhering moieties may confer increased thermalstability.

The cross-linkable and cross-linking activating moieties can be added inappropriate ratios to facilitate cross-linking. The ratio to be used maydepend on the cross-linkable and/or cross-linking activating moietiesused, the rate of cross-linking desired, and/or other reactionconditions appreciated by those of skill in the art. For example, aratio of at least about 1:1; at least about 1:2, at least about 1:5, atleast about 1:10; at least about 1:15, or at least about 1:20 may beused.

It will be recognized by those of skill in the art that certain of thesecross-linking activating moieties may be suitable for use alone, i.e.,without a corresponding cross-linkable moiety. For example, biotingroups, amine groups, carboxylic acid groups, cyanate groups (e.g.isothiocyanate), thiol groups, disulfide groups, cyano groups (e.g.,a-halocarbonyl groups, α, β-unsaturated carbonyl groups), an acetategroup (e.g., a-haloacetate group), hydrazine groups, cyanoacrylate,acrylic glue, and/or silicone moieties, as well as bifunctional linkers,may be used to bring about cross-linking of damaged lung tissue withoutthe use of a separate cross-linkable moiety. Further, variouscombinations of cross-linking activating moieties may be used,administered together at the same time or separately at different timesof administration. For instance, a dipolyaldehyde and/or polyaldehydemay be combined with a mixture of proteins, such as albumin and/orcollagen, and optionally other minor additives. Also, as mentionedabove, the cross-linking activating moiety may in some embodiments becoupled to an adhering moiety, for example, to an alpha-1 antitrypsinmolecule, fragment thereof, and/or derivative thereof; or to acombination of adhering moieties, including, for example, anycombination of types of adhering moieties provided herein.

It is also to be understood that some embodiments would not require across-linking activating moiety for initiation of cross-linking. Forexample, if fibrin is used as the cross-linkable moiety, e.g., a fibrinmonomer, such as fibrin I monomers, fibrin II monomers and/or des BBfibrin monomers, the monomers may spontaneously cross-link. Forinstance, fibrin I monomers may cross-link upon contacting a subject'sblood, which contains thrombin and factor XII.

Various types of cross-linking reactions may be used in the practice ofthe present invention including, for example, free radical reactions,cross-linking by zwitterions and/or ion pairs, anions and/or cations.See e.g., U.S. Pat. Nos. 6,010,714; 5,582,834; 5,575,997; 5,514,372;5,514,371 and 5,328,687 to Leung et al. and 5,981,621. Cross-linkingreactions of the present invention may also involve amide formation,imine formation, carbodiimide condensation, disulfide bond formation,and use of a specific binding pair, e.g., using a biotin-avidininteraction.

In some preferred embodiments, the method for reducing lung volume doesnot damage epithelial cells within lung tissues, e.g., it may not causescar tissue formation, and/or may not cause fibroblast proliferation,and/or may not cause collagen synthesis. In some preferred embodiments,the methods cross-link and/or seal sites of damaged lung tissue withinan alveolus, more preferably within an enlarged alveolus distal to aterminal bronchiole. In some preferred embodiments, the methods of thepresent invention do not cause occlusion of a lumen of a bronchial tubeof a lung of the subject. Without being limited to a particularmechanism, methods of the present invention can reduce lung volume bykeeping cross-linked and/or sealed enlarged air spaces, rather than by(mechanically) attempting to block air-flow to damaged lung tissue. Thatis, in preferred embodiments, cross-linking serves to keep collapsedand/or sealed blind ending sacs, rather than there being any or anysubstantial amount of lung tissue distal to the cross-linked sites. Inyet still preferred embodiments, the lung volume reducing methods of thepresent invention can be carried out without the use of open surgery,e.g., thoracotomy.

In some preferred embodiments, the method for reducing lung volume caninvolve damage to lung tissue. For example, in some embodiments asclerosing agent can be used as part of the administered gluecomposition, for instance, a sclerosing agent may be coupled to anadhering moiety of the present invention. In some embodiments, thesclerosing agent may be administered alone; or it may be administeredseparately at the same time as, before, or after administration ofadhering, cross-linkable, and/or cross-linking activating moieties ofthe present invention. The sclerosing agent can serve to bring aboutscar tissue formation, and/or fibroblast proliferation, and/or collagensynthesis, facilitating sealing of regions of damaged lung tissue.Sclerosing agents that may be used in the present invention includeDoxycycline, Bleomycin, Minocycline, Doxorubicin, Cisplatin+Cytarabine,Mitoxantrone, Corynebacterium Parvum, Streptokinase, Urokinase, and thelike. Other agents and/or methods for damaging lung tissue may also beused in the practice of the present invention, optionally along withcomponents of the extracellular matrix e.g., hyaluronic acid. See e.g.,U.S. Publication No. 2004/0047855.

In some embodiments, cross-linking activating moieties are administeredafter allowing sufficient time for adhering of the administeredcross-linkable moieties to lung tissue. In preferred embodiments, theadhering moiety adheres in at least about 30 seconds, at least about 1minute, at least about 3 minutes, or at least about 5 minutes. Inpreferred embodiments, the adhering moiety adheres in less than about 3hours, in less than about 2 hours, in less than about 1 hour, in lessthan about 45 minutes, in less than about 30 minutes, in less than about20 minutes, or in less than about 10 minutes. Also, in some embodiments,unbound adhering moiety may be removed from the lungs, e.g., by lavageand/or washing (e.g., with saline) and/or by collapsing, beforeadministration of cross-linking activating moiety.

Cross-linking and/or gluing may be facilitated by deflating and/orcollapsing a first portion or all of the lung of the subject, preferablywhere the first portion comprises a selected localized region of damagedlung tissue. Such deflating and/or collapsing can be achieved by anytechniques known in the art or herein disclosed. For example, thecollapsing may involve the use of negative pressure from within the lungand/or positive pressure from without the lung. Also, in someembodiments, a preparation to induce and/or facilitate collapse may beused, e.g., a physiologically acceptable solution containing ananti-surfactant, such as an agent that can increase surface tension offluids lining alveoli. For example, an anti-surfactant may beadministered prior to, during and/or after administration of thecomposition comprising a cross-linkable moiety and/or a cross-linkingactivating moiety. For instance, fribrin and/or fibrinogen may be used,which can act both as an anti-surfactant as well as aidingcross-linking.

Other suitable surfactants that may be used to facilitate cross-linkingand/or gluing include Triton x-100, beractant, colfosceril, and/orpalmitate; anionic surfactants such as sodium tetradecyl sulfate;cationic surfactants such as tetrabutylammonium bromide and/orbutyrylcholine chloride; nonionic surfactants such as polysorbate 20(e.g., Tween 20), polysorbate 80 (e.g. Tween 80), and/or poloxamers;amphoteric and/or zwitterionic surfactants such asdodecyldimethyl(3-sulfopropyl)ammonium hydroxide, inner salt; amines,imines and/or amides, such as arginine, imidazole, povidine, tryptamine,and/or urea; alcohols such as ascorbic acid, ethylene glycol, methylgallate, tannins and/or tannic acid; phosphines, phosphites andphosphonium salts, such as triphenylphosphine and/or triethyl phosphite;inorganic bases and/or salts, such as calcium sulfate, magnesiumhydroxide, sodium silicate, and/or sodium bisulfite; sulfur compoundssuch as polysulfides and/or thiourea; polymeric cyclic ethers such ascalixarenes, crown ethers, monensin, nonactin, and/or polymericepoxides; cyclic and acyclic carbonates; organometallics (e.g.,naphthenate and manganese acetylacetonate); phase transfer catalysts(e.g., Aliquat 336); and radical initiators and radicals (e.g.,di-t-butyl peroxide and/or azobisisobutyronitrile).

Cross-linking and/or gluing may also be facilitated by filling the lungor a portion thereof with an absorbable gas, such as oxygen, e.g., topromote atelectasis. Ingenito et al., “Bronchoscope volume reduction—Asafe and effective alternative to surgical therapy for emphysema,”American Journal of Respiratory and Critical Care Medicine, Vol 164 pgs295-301 (2001).

In some embodiments, a lavage of saline may be used to reduce the amountof surfactant naturally occurring in the lungs. Cross-linking and/orgluing may also be facilitated by use of a lavage capable of removing,e.g., any other moieties that may impede, reduce and/or otherwiseinterfere with adhering. For example, in some embodiments, cross-linkingmay be facilitated by use of an anti-secretory agent that hinders and/orprevents mucous secretion in the lung or a portion thereof. For example,the anti-secretory agent may be administered prior to, during, and/orafter administration of the glue composition comprising thecross-linkable moiety and/or the cross-linking activating moiety and/orother moiety. Examples of anti-secretory agents that may be usedinclude, for example, anticholinergic moieties, atronie, and/oratropinic moieties. Removal of mucous or excessive mucous from the lung,preferably from enlarged alveoli distal to terminal bronchioles, e.g.,by washing, can also facilitate cross-linking and/or gluing and/or theadhering of an adhering moiety to lung tissue. Adhesion of a gluecomposition to a mucous-coated wall within a bronchus, bronchiole, oralveolus can be facilitated by virtue of adhering moieties of thepresent invention adhering to lung tissue and, for example, reducingand/or avoiding slippage.

In some embodiments, mechanical force may be used externally to push onearea of the lung closer to another, for example, to help collapse and/ordeflate an enlarged air space. A portion of a lobe of the lung may bepressed externally using, for example, a balloon, air pressure, manualpressure, and/or an instrument such as a paddle, a net, a strap that canbe synched up, or magnets. In some embodiments, such pressure is appliedto two or more sides of a lung lobe simultaneously. For example,endoscopes and/or magnetic probes can be used to apply local pressure(applenate) to more than one side.

In some embodiments, a first portion or all of the lung may be drawntogether from the inside using, for example, a cable and hook to graband pull tissue, for instance, towards the user. Other devices that canbe used include graspers, such as an expanding grasper assembly that canbe sheathed; and/or anchors that can be left behind, for example, bybeing uncoupled from a cable or wire after lung tissue has been drawntogether. In some embodiments, magnetic probes can be placed atdifferent locations within the lung where the probes attract oneanother, thereby attracting one region of the lung to the other, e.g.,one bronchi to another. Additionally, mechanical force may be used tochange the shape of such devices after insertion, such as by using acore wire or activating a NiTi device after placement. In still otherembodiments, the lungs or a first portion thereof are deflatedtrans-thoracically. Other methods and/or devices known in the art tofacilitate lung deflation and/or collapse may also be employed, e.g. seeU.S. Publication No. 2003/0070682.

Such deflating and/or collapsing is preferably carried out afterallowing sufficient time for distribution of the administered gluecomposition to damaged lung tissue. In some embodiments, for example,deflating and/or collapsing is carried out approximately 2 toapproximately 3 minutes after administration. Also, the lung, or a firstportion thereof, is preferably allowed to remain in a collapsed and/ordeflated state for a time sufficient to permit cross-linking and/orgluing to take place, sealing segments of the lung to which the gluecomposition has been administered. Depending on the glue compositionused, e.g., the adhering moieties used, the lung or a first portionthereof can be kept deflated and/or collapsed for at least approximately3 days, at least approximately 2 days (48 hours), at least approximately24 hours, at least approximately 12 hours, at least approximately 5hours, at least approximately 1 hour, at least approximately 45 minutes,at least approximately 20 minutes, at least approximately 10 minutes, atleast approximately 5 minutes, at least approximately 2 minutes, atleast approximately 1 minute, at least approximately 30 seconds, or atleast approximately 15 seconds. In some embodiments, the lung or a firstportion thereof can be kept deflated and/or collapsed for less thanabout 30 minutes, less than about 20 minutes, less than about 10minutes, or less than about 0.8 minutes.

In some embodiments, a catalytic amount of a rate modifier may be addedto modify the rate of the cross-linking and/or gluing reaction. Forexample, various set or cure times may be used, where the cross-linkingreaction occurs in at least about 20 seconds, at least about 30 seconds,at least about 1 minute, at least about 90 seconds, at least about 2minutes, at least about 150 seconds, at least about 3 minutes, at leastabout 4 minutes, at least about 5 minutes, at least about 6 minutes, atleast about 10 minutes, or at least about 15 minutes. The cross-linkingreaction may occur in less than about 20 minutes, in less than about 25minutes, in less than about 30 minutes, in less than about 1 hour, inless than about 2 hours, or in less than about 3 hours. Cure times maybe tailored by use of various techniques known in the art, for example,by using buffers having different pH values.

A second portion of the lung can then be re-inflated, where the secondportion comprises part, but preferably not all, of the first portion orall of the lung that was deflated and/or collapsed. In preferredembodiments, this second portion does not comprise at least some damagedlung tissue, which remains collapsed and/or sealed by virtue of thecross-linking and/or gluing. The cross-linking and/or gluing preferablyforms a stable mesh that keeps the collapsed region from re-inflating.In more preferred embodiments, the majority of damaged lung tissueremains cross-linked and/or glued (and thereby collapsed), while themajority of non-damaged lung tissue is left in a functional condition.For example, at least about 60%, at least about 80%, and most preferablyat least about 90% of damaged lung tissue is cross-linked and/or glued;while less than about 40%, less than about 20%, and most preferably lessthan about 10% of non-damaged lung tissue remains not cross-linkedand/or not glued. Reduction in overall lung volume improves mechanicalfunction, e.g., mechanical functioning of healthier and/or more elastictissue.

In preferred embodiments, cross-linking and/or gluing results in atleast about a 0.5% overall lung volume reduction, at least about a 1%overall lung volume reduction, at least about a 1.5% overall lung volumereduction, at least about a 2% overall lung volume reduction, at leastabout a 3% overall lung volume reduction, at least about a 4% overalllung volume reduction, at least about a 5% overall lung volumereduction, or at least about a 10% overall lung volume reduction. Inpreferred embodiments, cross-linking and/or gluing results in less thanabout a 40%, less than about a 35%, less than about a 30%, less thanabout a 25%, less than about a 20%, or less than about a 15% overalllung volume reduction. Such reduction may be achieved upon a single ormultiple administrations of compositions of the present invention. Areduction of about 2% to about 3% overall lung volume reduction can beexpected to produce a beneficial effect in a subject receiving suchtreatment, e.g., at least to a similar extent as that produced in LVRS.

Also in preferred embodiments, the cross-linking and/or gluing ispermanent, or at least semi-permanent, for a period of time betweensuccessive treatments as described herein, e.g., resistingbiodegradation (e.g., hydrolysis) for the period of time betweenadministrations of a glue composition of the present invention. Incertain embodiments, at least about 70%, at least about 80%, at leastabout 90%, or at least about 98% of the cross-links and/or glue remainintact for a period of time. In some preferred embodiments, the periodis at least about one month, at least about 2 months, at least about 3months, at least about 6 months, at least about a year, at least about 2years, at least about 3 years, at least about 5 years, or at least about10 years. In some preferred embodiments, the period is less than about50 years, less than about 30 years, less than about 20 years, or lessthan about 15 years. In most preferred embodiments, the cross-linkingand/or gluing keeps some damaged lung tissue collapsed and/or sealed forthe remainder of the life of the subject, for example, resistingbiodegradation indefinitely.

One of skill in the art will recognize that the permanence and/orbiodegradability of the cross-links and/or glue can depend on thecross-linkable moiety, the cross-linking activating moiety, and/or theconditions of cross-linking and/or other agents and/or moieties used,and can be controlled accordingly, e.g., by techniques known the artand/or disclosed herein.

In preferred embodiments, some or all of the cross-links and/or glue arestrong enough to withstand mechanical pressures experienced within thelung. For example, the strain range corresponding to functional residualcapacity during normal breathing does not result in breakage of at leastabout 30%, at least about 40%, at least about 50%, at least about 60%,at least about 70%, at least about 80%, at least about 90%, or at leastabout 95% of the cross-links and/or glue in some preferred embodiments.

In some preferred embodiments, the cross-links and/or glue exhibit atear strength of at least about 50 g/sq. cm, at least about 100 g/sq.cm, at least about 200 g/sq. cm, or at least about 300 g/sq. cm. In somepreferred embodiments, the cross-links and/or glue exhibit a tearstrength of 5,000 g/sq. cm, less than about 3,000 g/sq. cm, less thanabout 1500 g/sq. cm, less than about 1300 g/sq. cm, less than about1200/g/sq. cm, less than about 1000 g/sq. cm, less than about 800 g/sq.cm, less than about 600 g/sq. cm, or less than about 400 g/sq. cm.

Similarly, in preferred embodiments, the binding interaction between anadhering moiety and lung tissue is permanent, or at leastsemi-permanent, for a period of time between successive treatments asdescribed herein, e.g., binding irreversibly, substantiallyirreversibly, or at least with a high binding constant, e.g., to resistdissociation for the period of time between administrations of a gluecomposition of the present invention. For example, an alpha-1antitrypsin moiety may form a pseudo-irreversible equimolar complex withneutrophil elastase in some embodiments. See, e.g., Sifers et al.,“Genetic Control of Human Alpha-1 Antitrypsin”, Mol. Biol. Med., Vol. 6pgs. 127-135 (1989). Without being limited to a particular theory ormode of action, the alpha-1 antitrypsin moiety may form an acyl-enzymecomplex with an elastase component of lung tissue. In some embodiments,binding can be further enhanced by genetic modification or by shufflingof known binding domains. As another example, a serpin moiety may reactwith its corresponding protease to form a sodium dodecylsulfate(SDS)-stable equimolar complex. Without being limited to aparticular theory or mode of action, the complex between a serpin andits corresponding protease may involve a covalent ester bond linkage,where an active site Serine residue of the protease binds a C-terminalresidue of a cleaved form of the serpin to form an acyl-enzyme complex.See, e.g., U.S. Publication No 2003/0216321. As yet another example, amonocyte elastase inhibitor moiety can form a covalent complex and/or anessentially irreversible complex with elastase. See, e.g., InternationalPublication WO 96/10418 and U.S. Pat. No. 5,827,672.

In certain embodiments, at least about 70%, at least about 80%, at leastabout 90%, or at least about 98% of the adhering moieties remain boundto lung tissue for a period of time. In some preferred embodiments, theperiod is at least about one month, at least about 2 months, at leastabout 3 months, at least about 6 months, at least about a year, at leastabout 2 years, at least about 3 years, at least about 5 years, or atleast about 10 years. In some preferred embodiments, the period is lessthan about 50 years, less than about 30 years, less than about 20 years,or less than about 15 years. In most preferred embodiments, the bindingkeeps some damaged lung tissue collapsed and/or sealed for the remainderof the life of the subject, for example, resisting dissociationindefinitely.

FIG. 1 a illustrates one embodiment of a method to reduce lung volumeusing a glue composition comprising a cross-linkable moiety coupled toan adhering moiety that adheres to lung tissue. This figure provides anoverview only, and is in no way intended to be limiting with respect tothe present invention. For example, those skilled in the art willreadily appreciate variations and modifications of the schemeillustrated. FIG. 1 a schematically illustrates a bronchoscope placed ina bronchus from which a catheter extends to a segmental and subsegmentalbronchus. The catheter features a distended balloon-like structure nearits distal tip. The balloon-like structure anchors the catheter withinthe subsegmental bronchus, positioning it for delivery of a gluecomposition of the present invention in aerosol and/or non-aerosol formto a selected localized region of damaged lung tissue.

FIG. 1 b schematically illustrates a terminal bronchiole, terminating inthe airspace of an alveolus. The figure also illustrates a component oflung tissue found within the walls of the air space and/or within theepithelial lining fluid. As mentioned above, the airspace may beover-inflated and/or enlarged in certain pulmonary conditions, such asemphysema.

A glue composition of the invention is administered, where the gluecomposition comprises a cross-linkable moiety (X) coupled to an adheringmoiety that can adhere to lung tissue, such as a component of lungtissue. FIG. 1 b illustrates how different adhering moieties adhere tolung tissue components at various sites within the air space.

Following collapse and cross-linking, the cross-linkable moieties becomecross-linked, for example, via a cross-linking activating moiety. Thecross-linking activating moiety may comprise a di-functional group,depicted in the figure as —Y—R—Y—, where Y represents a group capable ofcoupling to the cross-linkable moieties (X), e.g., to form covalentlinkages between two cross-linkable moieties, and R represents a linkingmoiety between the Y groups, for example, but not limited to, analiphatic chain. The cross-linking activating moiety couples thecross-linkable moieties that are themselves coupled to adhering moietiesbound to lung tissue components found at various sites within the airspace. FIG. 1 b illustrates how cross-linking can keep the walls of theair space closer together even after re-inflation of the lung, therebyreducing overall lung volume.

The methods of reducing lung volume described herein find use in thetreatment of a number of pulmonary conditions in animal subjects. Theterm “animal subject” as used herein includes humans as well as othermammals. The term “treating” as used herein includes achieving atherapeutic benefit and/or a prophylactic benefit. By therapeuticbenefit is meant eradication or amelioration of the underlying pulmonarycondition being treated. For example, in an emphysematous patient,therapeutic benefit includes eradication or amelioration of theunderlying emphysema, including improved lung function, exercisecapacity, quality of life, and reduced hospitalization. Also, atherapeutic benefit is achieved with the eradication or amelioration ofone or more of the physiological symptoms associated with the underlyingpulmonary condition such that an improvement is observed in the subject,notwithstanding the fact that the subject may still be afflicted withthe pulmonary condition. For example, with respect to emphysema,administration of compositions of the invention can provide therapeuticbenefit not only when areas lacking elasticity are collapsed, but alsowhen an improvement is observed in the subject with respect to otherdisorders that accompany emphysema like chronic pulmonary infection. Forexample, addition of adhering moieties comprising protease inhibitorsmay ameliorate emphysema by reducing protease activity, e.g., asdescribed in the art. For prophylactic benefit, a glue composition ofthe present invention may be administered to a subject at risk ofdeveloping a pulmonary condition, for example, emphysema, or to asubject reporting one or more of the physiological symptoms of such acondition, even though a diagnosis may not have been made.

Another aspect of the present invention provides a method of reducinglung volume by providing a glue composition comprising a first adheringmoiety and a second adhering moiety wherein said adhering moieties arecoupled and wherein said adhering moieties adhere lung tissue;administering said glue composition to a localized region of damagedlung tissue of a subject; collapsing a first portion or all of the lungof said subject wherein said first portion comprises said localizedregion of damaged lung tissue; allowing said adhering moieties to adheredifferent sites of lung tissue, and re-inflating a second portion of thelung of said subject wherein said second portion does not comprise saiddamaged lung tissue, thereby reducing lung volume.

In preferred embodiments, the different sites comprise different siteswithin an enlarged air space, e.g., within alveolar walls of anover-inflated alveolus distal to a terminal bronchiole, ascharacteristic of some pulmonary conditions, including emphysema. Forexample, the first adhering moiety can adhere to a first component oflung tissue and the second adhering moiety can adhere to a secondcomponent of lung tissue, where the first and second components occur atdifferent sites. As the coupled adhering moieties bind to differentsites within an air space, following deflation and/or collapse, thecoupled adhering moieties can act to keep different sites closertogether, thereby keeping the air space in a collapsed and/or sealedstate. Also regions of damaged lung tissue can be selectively collapsedand/or sealed by administering a glue composition of the presentinvention to a selected localized region of damaged lung tissue,preferably freeing up space for the expansion of remaining non-damagedor healthier tissue.

In some preferred embodiments, the method is performed with prioridentification of the damaged lung tissue. For example, the lungs of thesubject may be imaged to identify regions or sites of damaged tissuebefore administering a glue composition of the invention to the subject.Such identification may involve any techniques known in the art, to bedeveloped, described herein, and/or described in U.S. nonprovisionalapplications entitled “Targeting Damaged Lung Tissue UsingCompositions,” filed Dec. 8, 2004; “Targeting Damaged Lung Tissue,”filed Dec. 8, 2004; “Targeting Sites of Damaged Lung Tissue UsingComposition,” filed Dec. 8, 2004; “Targeting Sites of Damaged LungTissue,” filed Dec. 8, 2004; “Imaging Damaged Lung Tissue UsingCompositions,” filed Dec. 8, 2004; “Imaging Damaged Lung Tissue,” filedDec. 8, 2004; “Glue Compositions for Lung Volume Reduction,” filed Dec.8, 2004; “Lung Volume Reduction Using Glue Compositions,” filed Dec. 8,2004; and “Lung Volume Reduction Using Glue Composition,” filed Dec. 8,2004, each of which is herein incorporated in its entirely thatfacilitate identification of regions of damaged lung tissue. Regions ofdamaged lung tissue include areas of the lung affected by a pulmonarycondition or that are affected to a greater extent compared with other,healthier areas of the lung. In emphysema, for example, such regions caninclude regions featuring “blebs,” that is, regions where progressivedestruction of elastic tissue has caused loss of lung recoil andconsequent hyper-extension.

Current techniques that may be used to identify damaged lung tissue inthe present invention include radiology (e.g., chest X-rays) and CTscans. For example, review of CT scans of the chest, preferablyhigh-resolution CT scans, can indicate localized regions of damaged lungtissue that may be selected for volume reduction.

Because the collapsed tissue occupies a smaller volume than the enlargedalveoli at sites of damaged tissue, methods of this invention can reducelung volume overall. The present invention can thus provide anon-surgical, less-invasive and/or safer approach for achieving at leastsome of the benefits of lung volume reduction surgery. Further,providing the glue composition to a localized region of damaged lungtissue allows localized volume reduction, which in turn minimizesuntoward side effects, such as exacerbating V/Q imbalance, changingarterial oxygenation, or triggering acute hypoxemia. Ingenito et al.,“Bronchoiscopic Lung Volume Reduction Using tissue engineeringprinciples”, American Journal of Respiratory and Critical Care Medicine,Vol. 167 pgs. 771-778 (2002). It is to be understood also that themethods of the present invention may be used in conjunction with asurgical procedure, such as LVRS, as well as other approaches fortreating pulmonary conditions, including cross-linking and/or gluingmethods described herein, and/or other methods described in any of theapplications entitled “Targeting Damaged Lung Tissue UsingCompositions,” filed Dec. 8, 2004; “Targeting Damaged Lung Tissue,”filed Dec. 8, 2004; “Targeting Sites of Damaged Lung Tissue UsingComposition,” filed Dec. 8, 2004; “Targeting Sites of Damaged LungTissue,” filed Dec. 8, 2004; “Imaging Damaged Lung Tissue UsingCompositions,” filed Dec. 8, 2004; “Imaging Damaged Lung Tissue,” filedDec. 8, 2004; “Glue Compositions for Lung Volume Reduction,” filed Dec.8, 2004; “Lung Volume Reduction Using Glue Compositions,” filed Dec. 8,2004; and “Lung Volume Reduction Using Glue Composition,” filed Dec. 8,2004, each of which is herein incorporated in its entirely.

In some preferred embodiments, the method for reducing lung volume doesnot damage epithelial cells within lung tissues, e.g., it may not causescar tissue formation, and/or may not cause fibroblast proliferation,and/or may not cause collagen synthesis. In some preferred embodiments,the methods cross-link and/or seal sites of damaged lung tissue withinan alveolus, more preferably within an enlarged alveolus distal to aterminal bronchiole. In some preferred embodiments, the methods of thepresent invention do not cause occlusion of a lumen of a bronchial tubeof a lung of the subject. Without being limited to a particularmechanism, methods of the present invention can reduce lung volume bysealing enlarged air spaces, rather than by (mechanically) attempting toblock air-flow to damaged lung tissue. That is, in preferredembodiments, administering a glue composition to a localized region ofdamaged lung tissue serves to seal and/or keep collapsed blind endingsacs, rather than there being any or any substantial amount of lungtissue distal to the collapsed region. In yet still preferredembodiments, the lung volume reducing methods of the present inventioncan be carried out without the use of open surgery, e.g., thoracotomy.

In some preferred embodiments, the method for reducing lung volume caninvolve damage to lung tissue. For example, in some embodiments asclerosing agent can be used as part of the administered gluecomposition, for instance, a sclerosing agent may be coupled to anadhering moiety of the present invention. In some embodiments, thesclerosing agent may be administered alone; or it may be administeredseparately at the same time as, before, or after administration ofadhering moieties of the present invention. The sclerosing agent canserve to bring about scar tissue formation, and/or fibroblastproliferation, and/or collagen synthesis, facilitating sealing ofregions of damaged lung tissue. Sclerosing agents that may be used inthe present invention include Doxycycline, Bleomycin, Minocycline,Doxorubicin, Cisplatin+Cytarabine, Mitoxantrone, Corynebacterium Parvum,Streptokinase, Urokinase, and the like. Other agents and/or methods fordamaging lung tissue may also be used in the practice of the presentinvention, optionally along with components of the extracellular matrix,e.g., hyaluronic acid. See e.g., U.S. Publication No. 2004/0047855.

Collapse of lung tissue, e.g., collapse of an enlarged air spaces withinwhich a glue composition of the present invention adheres, may involvedeflating and/or collapsing a first portion or all of the lung of thesubject, preferably where the first portion comprises the selectedlocalized region of damaged lung tissue. Such collapsing can be achievedby any techniques known in the art or herein disclosed. For example, thedeflating and/or collapsing may involve the use of negative pressurefrom within the lung and/or positive pressure from without the lung.Also, in some embodiments, a preparation to induce and/or facilitatedeflation and/or collapse may be used, e.g., a physiologicallyacceptable solution containing an anti-surfactant, such as an agent thatcan increase surface tension of fluids lining alveoli. For example, ananti-surfactant may be administered prior to, during and/or afteradministration of the composition comprising coupled adhering moieties.For instance, fribrin and/or fibrinogen may be used. In someembodiments, a lavage of saline may be used to reduce the amount ofsurfactant naturally occurring in the lungs. Other suitable surfactantsthat may be used to facilitate collapse and/or deflation include Tritonx-100, beractant, colfosceril, and/or palmitate; anionic surfactantssuch as sodium tetradecyl sulfate; cationic surfactants such astetrabutylammonium bromide and/or butyrylcholine chloride; nonionicsurfactants such as polysorbate 20 (e.g., Tween 20), polysorbate 80(e.g. Tween 80), and/or poloxamers; amphoteric and/or zwitterionicsurfactants such as dodecyldimethyl(3-sulfopropyl)ammonium hydroxide,inner salt; amines, imines and/or amides, such as arginine, imidazole,povidine, tryptamine, and/or urea; alcohols such as ascorbic acid,ethylene glycol, methyl gallate, tannins and/or tannic acid; phosphines,phosphites and phosphonium salts, such as triphenylphosphine and/ortriethyl phosphite; inorganic bases and/or salts, such as calciumsulfate, magnesium hydroxide, sodium silicate, and/or sodium bisulfite;sulfur compounds such as polysulfides and/or thiourea; polymeric cyclicethers such as calixarenes, crown ethers, monensin, nonactin, and/orpolymeric epoxides; cyclic and acyclic carbonates; organometallics(e.g., naphthenate and manganese acetylacetonate); phase transfercatalysts (e.g., Aliquat 336); and radical initiators and radicals(e.g., di-t-butyl peroxide and/or azobisisobutyronitrile).

Deflation and/or collapse may also be facilitated by use of a lavagecapable of removing any other moieties that may impede, reduce and/orotherwise interfere with adhering. For example, in some embodiments,cross-linking may be facilitated by use of an anti-secretory agent thathinders and/or prevents mucous secretion in the lung or a portionthereof. For example, the anti-secretory agent may be administered priorto, during, and/or after administration of the glue compositioncomprising coupled adhering moieties. Examples of anti-secretory agentsthat may be used include, for example, anticholinergic moieties,atronie, and/or atropinic moieties. Removal of mucous or excessivemucous from the lung, preferably from enlarged alveoli distal toterminal bronchioles, e.g., by washing, can also facilitate binding ofthe coupled adhering moieties to lung tissue. Adhesion of a compositionof the present invention to a mucous-coated wall within a bronchus,bronchiole, or alveolus can be facilitated by virtue of adheringmoieties of the present invention adhering components of lung tissueand, e.g., reducing and/or avoiding slippage.

In some embodiments, mechanical force may be used externally to push onearea of the lung closer to another, for example, to help collapse anenlarged air space. A portion of a lobe of the lung may be pressedexternally using, for example, a balloon, air pressure, manual pressure,and/or an instrument such as a paddle, a net, a strap that can besynched up, or magnets. In some embodiments, such pressure is applied totwo or more sides of a lung lobe simultaneously. For example, endoscopesand/or magnetic probes can be used to apply local pressure (applenate)to more than one side.

In some embodiments, a first portion or all of the lung may be drawntogether from the inside using, for example, a cable and hook to graband pull tissue, for instance, towards the user. Other devices that canbe used include graspers, such as an expanding grasper assembly that canbe sheathed; and/or anchors that can be left behind, for example, bybeing uncoupled from a cable or wire after lung tissue has been drawntogether. In some embodiments, magnetic probes can be placed atdifferent locations within the lung where the probes attract oneanother, thereby attracting one region of the lung to the other, e.g.,one bronchi to another. Additionally, mechanical force may be used tochange the shape of devices after insertion, such as by using a corewire or activating a NiTi device after placement. In still otherembodiments, the lungs or a first portion thereof are deflatedtrans-thoracically. Other methods and/or devices known in the art tofacilitate lung collapse may also be employed, e.g. see U.S. PublicationNo. 2003/0070682.

Such deflation and/or collapsing is preferably carried out afterallowing sufficient time for distribution of the administered gluecomposition to a selected localized region of damaged lung tissue. Insome embodiments, for example, deflation and/or collapse is carried outapproximately 2 to approximately 3 minutes after administration of aglue composition of the present invention. Also, the lung, or a firstportion thereof, is preferably allowed to remain in a deflated and/orcollapsed state for a time sufficient to permit adhering of more thanone of the coupled adhering moieties to lung tissue components atdifferent sites of lung tissue in localized regions to which the gluecomposition has been administered. Depending on the glue compositionused, e.g., the adhering moieties used, the lung or a first portionthereof can be kept deflated and/or collapsed for at least approximately3 days, at least approximately 2 days (48 hours), at least approximately24 hours, at least approximately 12 hours, at least approximately 5hours, at least approximately 1 hour, at least approximately 45 minutes,at least approximately 20 minutes, at least approximately 10 minutes, atleast approximately 5 minutes, at least approximately 2 minutes, atleast approximately 1 minute, at least approximately 30 seconds, or atleast approximately 15 seconds. In some embodiments, the lung or a firstportion thereof can be kept deflated and/or collapsed for less thanabout 30 minutes, less than about 20 minutes, less than about 10minutes, or less than about 8 minutes.

A second portion of the lung can then be re-inflated, where the secondportion comprises part, but preferably not all, of the first portion orall of the lung that was deflated and/or collapsed. In preferredembodiments, this second portion does not comprise at least some damagedlung tissue, which remains collapsed and/or sealed by virtue of coupledadhering moieties bound to different sites of damaged lung tissue. Theadhering preferably keeps the collapsed region from re-inflating. Inmore preferred embodiments, the majority of damaged lung tissue remainscollapsed and/or sealed, while the majority of non-damaged lung tissueis left in a functional condition. For example, at least about 60%, atleast about 80%, and most preferably at least about 90% of damaged lungtissues is collapsed; while less than about 40%, less than about 20%,and most preferably less than about 10% of non-damaged lung tissue isnot and/or re-inflates. Reduction in overall lung volume improvesmechanical function, e.g., mechanical functioning of healthier and/ormore elastic tissue.

In preferred embodiments, binding of coupled adhering moieties resultsin at least about a 0.5% overall lung volume reduction, at least about a1% overall lung volume reduction, at least about a 1.5% overall lungvolume reduction, at least about a 2% overall lung volume reduction, atleast about a 3% overall lung volume reduction, at least about a 4%overall lung volume reduction, at least about a 5% overall lung volumereduction, at least about a 10% overall lung volume reduction. Inpreferred embodiments binding of coupled adhering moieties results inless than about a 40%, less than about a 35%, less than about a 30%,less than about a 25%, less than about a 20%, or less than about a 15%overall lung volume reduction. Such reduction may be achieved upon asingle or multiple administrations of glue compositions of the presentinvention. A reduction of about 2% to about 3% overall lung volumereduction can be expected to produce a beneficial effect in a subjectreceiving such treatment, e.g., at least to a similar extent as thatproduced in LVRS.

Also in preferred embodiments, the coupling between adhering moieties ispermanent or at least semi-permanent for a period of time betweensuccessive treatments as described herein, e.g., resistingbiodegradation (e.g., hydrolysis) for the period of time betweenadministrations of a glue composition of the present invention. Incertain embodiments, at least about 70%, at least about 80%, at leastabout 90%, or at least about 98% of the coupling between adheringmoieties remains intact for a period of time. In some preferredembodiments, the period is at least about one month, at least about 2months, at least about 3 months, at least about 6 months, at least abouta year, at least about 2 years, at least about 3 years, at least about 5years, or at least about 10 years. In some preferred embodiments, theperiod is less than about 50 years, less than about 30 years, less thanabout 20 years, or less than about 15 years. In most preferredembodiments, the coupled adhering moieties keep some damaged lung tissuecollapsed and/or sealed for the remainder of the life of the subject,for example, resisting biodegradation indefinitely. One of skill in theart will recognize that the permanence and/or biodegradability of thecoupling between adhering moieties can depend on the coupling techniquechosen and/or the coupling moiety used.

In preferred embodiments, some or all of the coupling moieties arestrong enough to withstand mechanical pressures experienced within thelung. For example, the strain range corresponding to functional residualcapacity during normal breathing does not result in breakage of at leastabout 30%, at least about 40%, at least about 50%, at least about 60%,at least about 70%, at least about 80%, at least about 90%, or at leastabout 95% of the coupling moieties in some preferred embodiments.

In some preferred embodiments, the coupling moieties exhibit a tearstrength of at least about 50 g/sq. cm, at least about 100 g/sq. cm, atleast about 200 g/sq. cm, or at least about 300 g/sq. cm. In somepreferred embodiments, the coupling moieties exhibit a tear strength ofless than about 5,000 g/sq. cm, less than about 3,000 g/sq. cm, lessthan about 1500 g/sq. cm, less than about 1300 g/sq/cm, less than about1200 g/sq. cm, less than about 1000 g/sq. cm, less than about 800 g/sq.cm, less than about 600 g/sq. cm, or less than about 400 g/sq. cm.

Similarly, in preferred embodiments, the binding between adheringmoieties and lung tissue is permanent or at least semi-permanent for aperiod of time between successive treatments as described herein, e.g.,binding irreversibly, substantially irreversibly, or at least with ahigh binding constant to resist dissociation for the period of timebetween administrations of a glue composition of the present invention.For example, an alpha-1 antitrypsin moiety may form apseudo-irreversible equimolar complex with neutrophil elastase in someembodiments. See, e.g., Sifers et al., “Genetic Control of Human Alpha-1Antitrypsin”, Mol. Biol. Med., Vol. 6 pgs. 127-135 (1989). Without beinglimited to a particular theory or mode of action, the alpha-1antitrypsin moiety may form an acyl-enzyme complex with an elastasecomponent of lung tissue. In some embodiments, binding can be furtherenhanced by genetic modification or by shuffling of known bindingdomains. As another example, a serpin moiety may react with itscorresponding protease to form a sodium dodecyl sulfate(SDS)-stableequimolar complex. Without being limited to a particular theory or modeof action, the complex between a serpin and its corresponding proteasemay involve a covalent ester bond linkage, where an active site Serineresidue of the protease binds a C-terminal residue of a cleaved form ofthe serpin to form an acyl-enzyme complex. See, e.g., U.S. PublicationNo. 2003/0216321. As yet another example, a monocyte elastase inhibitormoiety can form a covalent complex and/or an essentially irreversiblecomplex with elastase. See, e.g., International Publication WO 96/10418and U.S. Pat. No. 5,827,672.

In certain embodiments, at least about 70%, at least about 80%, at leastabout 90%, or at least about 98% of the adhering moieties remain boundto lung tissue for a period of time. In some preferred embodiments, theperiod is at least about one month, at least about 2 months, at leastabout 3 months, at least about 6 months, at least about a year, at leastabout 2 years, at least about 3 years, at least about 5 years, or atleast about 10 years. In some preferred embodiments, the period is lessthan about 50 years, less than about 30 years, less than about 20 years,or less than about 15 years. In most preferred embodiments, the adheringkeeps some damaged lung tissue collapsed and/or sealed for the remainderof the life of the subject, for example, resisting dissociationindefinitely.

FIG. 2 illustrates one embodiment of a method to reduce lung volumeusing a glue composition comprising coupled adhering moieties. Thisfigure provides an overview only, and is in no way intended to belimiting with respect to the present invention. For example, thoseskilled in the art will readily appreciate variations and modificationsof the scheme illustrated. FIG. 2 a schematically illustrates abronchoscope placed in a bronchus from which a catheter extends to asegmental and subsegmental bronchus. The catheter features a distendedballoon-like structure near its distal tip. The balloon-like structureanchors the catheter within the subsegmental bronchus, positioning itfor delivery of a glue composition of the present invention in aerosoland/or non-aerosol form to a selected localized region of damaged lungtissue.

FIG. 2 b schematically illustrates a terminal bronchiole, terminating inthe airspace of an alveolus. The figure also illustrates lung tissuecomponents found within the walls of the airspace and/or within theepithelial lining fluid. As mentioned above, the air space may beover-inflated and/or enlarged in certain pulmonary conditions, such asemphysema.

A glue composition of the invention is administered, where the gluecomposition comprises adhering moieties that are coupled, for example,via a coupling moiety. FIG. 2 b illustrates how, followingadministration, one of the adhering moieties can adhere to a lung tissuecomponent within the air space.

FIG. 2 b also illustrates how the two adhering moieties can adhere tolung tissue components at two different sites within the air space.Following deflation, the walls of the alveolus are brought into closerproximity, allowing the second adhering moiety to adhere to a lungtissue component at a different site within the air space. The bindingof coupled adhering moieties to hitherto further-apart sites of lungtissue serves to help keep the walls of the air space closer together. Apreviously enlarged and/or distended alveolus may thus be kept in acollapsed and/or sealed state after re-inflation, thereby reducingoverall lung volume.

Glue compositions of the present invention may also comprise an imagingmoiety, for example, an imaging moiety coupled to an adhering moiety, across-linkable moiety, a cross-linking moiety and/or a sclerosing agentused. An imaging moiety may assist in non-intrusive visualization and/ormonitoring of the collapsed and/or sealed lung tissue. For example,imaging moieties can afford detection of sealed damaged lung tissue andpreferably facilitate monitoring of the presence, position, extent,and/or degradation of the cross-links, coupling moieties and/or glue,and/or dissociation of the adhering moiety.

The imaging moiety may be imaged by any methods known in the art and/ordescribed herein. For example, imaging may be carried out viatraditional radiological techniques, including, for example the use ofan X-ray, computer tomography (CT), and/or the use of more advancedtechniques such as a positron emission tomography (PET) scan, nuclearscans, and/or scintigraphy, as well as magnetic resonance imaging (MRI),functional magnetic resonance imaging (FMRI), magnetoencephalography(MEG), and single photon emission computerized tomography (SPECT). Suchimaging techniques can be used to detect localized imaging moieties invitro or in vivo, preferably in vivo. High resolution scans, e.g., ahigh resolution CT scan, are preferable. In more preferred embodiments,such imaging produces a detailed map of the lungs, showing sites ofglued and/or sealed tissue and/or the extent of collapse, e.g., by theconcentration of imaging moieties at localized sites within the lungs towhich the glue composition was administered.

The method of detection used may depend on the imaging moietyadministered. For example, ultrasound imaging can be used to detect anechogenic imaging moiety and/or an imaging moiety capable of generatingan echogenic signal and/or other ultrasound imaging moieties. X-ray canbe used to detect a heavy atom imaging moiety (e.g., having atomicweight of about 38 or above). Light imaging can be used to detect animaging moiety capable of scattering and/or absorbing and/or emittinglight. MR imaging can be used to detect an imaging moiety comprising anon-zero nuclear spin isotope (such as F-19) and/or an imaging moietyhaving unpaired electron spins. PET, scintigraphy, and/or SPECT can beused to detect a radionuclide imaging moiety.

For example, in some embodiments, an imaging moiety comprising aradioactive gamma emitter can be used, and can be detected via a gammacamera, scintillation counter, and/or other device capable of detectinggamma radiation. Radiation imaging cameras can use a conversion mediumto absorb high-energy gamma rays and displace an electron, which emits aphoton on its return a lower orbital state. Some cameras also usephotoelectric detectors, e.g., arranged in a spatial detection chamberto determine the position of an emitted photon, as well as circuitry toanalyze the photons detected in the chamber to help produce an image.

In embodiments using an imaging moiety comprising a magnetic species,e.g., a paramagnetic atom, the imaging moiety can be detected by MRimaging, e.g., a magnetic resonance imaging system can be used. In suchsystems, a strong magnetic field can be used to align nuclear spinvectors of atoms, such as paramagnetic atoms at localized sites of lungtissue. The field can then be distributed by the paramagnetic atoms atsuch sites. As the nuclei return to equilibrium alignments, an image oflung tissue, e.g., localized sites of collapsed and/or sealed lungtissue, can be obtained.

Some embodiments of the present invention employ both imaging andvolume-reducing aspects of the invention described herein. In someembodiments, the imaging moiety may be coupled to an adhering moietythat itself is coupled to a cross-linkable moiety and/or one or moreother adhering moieties. In some embodiments, a second compositioncomprising an adhering moiety coupled to an imaging moiety can be used.In some embodiments, lung volume reduction, e.g., using gluecompositions and/or methods described herein, may be preceded and/orfollowed by imaging, e.g., and the images compared, e.g., to determinethe extent of collapse and/or sealing achieved in regions of selecteddamaged lung tissue.

Administration of a glue composition comprising an adhering moietycoupled to all and any of an imaging moiety, a cross-linkable moiety, across-linking activating moiety, other adhering moiety, and/or othermoiety and/or agent, may be followed by washing. The term “washing” asused herein refers to administration of a washing moiety that canfacilitate removal of an adhering moiety from lung tissue. For instance,a washing step may follow administration and imaging of a gluecomposition comprising an adhering moiety coupled to an imaging moietyto free up sites. Following washing, a glue composition comprising anadhering moiety coupled to a cross-linkable moiety and/or coupled toanother adhering moiety may be administered to the subject, for exampleto achieve lung volume reduction by methods described herein. Washingmoieties suitable for use in the present invention include, for example,soluble components of lung tissue to which adhering moieties can bind.The soluble components can compete with lung tissue components forbinding with the adhering moieties. Preferably, the soluble componentsare modified so as to reduce and/or eliminate undesirable propertiesbefore administration to a subject. For example, a mutant elastasepolypeptide may be used that can still bind to alpha-1 antitrypsin butthat cannot degrade lung tissue or degrades lung tissue to a lesserextent than non-mutant elastase.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention, and it should beunderstood that various alternatives to the embodiments of the inventiondescribed herein may be employed in practicing the invention. It isintended that the following claims define the scope of the invention andthat methods and compositions within the scope of these claims, alongwith their equivalents, are covered thereby.

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent or patent application wasspecifically and individually indicated as being incorporated byreference.

1. A composition for sealing lung tissue, comprising: a bifunctionalchemical linker; a first adhering moiety connected via a first amidebond linkage to a first site on the bifunctional chemical linker; and asecond adhering moiety connected via a second amide bond linkage to asecond site on the bifunctional chemical linker, wherein the first andsecond adhering moieties adhere to different sites bearing elastase in alung, and each adhering moiety comprises a member selected from thegroup consisting of an alpha-1 antitrypsin, an elafin, and a serpin.2-22. (canceled)